Multimodal silica-based nanoparticles

ABSTRACT

The present invention provides a fluorescent silica-based nanoparticle that allows for precise detection, characterization, monitoring and treatment of a disease such as cancer. The nanoparticle has a range of diameters including between about 0.1 nm and about 100 nm, between about 0.5 nm and about 50 nm, between about 1 nm and about 25 nm, between about 1 nm and about 15 nm, or between about 1 nm and about 8 nm. The nanoparticle has a fluorescent compound positioned within the nanoparticle, and has greater brightness and fluorescent quantum yield than the free fluorescent compound. The nanoparticle also exhibits high biostability and biocompatibility. To facilitate efficient urinary excretion of the nanoparticle, it may be coated with an organic polymer, such as poly(ethylene glycol) (PEG). The small size of the nanoparticle, the silica base and the organic polymer coating minimizes the toxicity of the nanoparticle when administered in vivo. In order to target a specific cell type, the nanoparticle may further be conjugated to a ligand, which is capable of binding to a cellular component associated with the specific cell type, such as a tumor marker. In one embodiment, a therapeutic agent may be attached to the nanoparticle. To permit the nanoparticle to be detectable by not only optical fluorescence imaging, but also other imaging techniques, such as positron emission tomography (PET), single photon emission computed tomography (SPECT), computerized tomography (CT), bioluminescence imaging, and magnetic resonance imaging (MRI), radionuclides/radiometals or paramagnetic ions may be conjugated to the nanoparticle.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/215,879, filed Mar. 17, 2014, which claims priority to U.S.Provisional Application No. 61/794,414, filed Mar. 15, 2013. Thedisclosures of each of the above-referenced applications areincorporated by referenced herein in their entireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under grant numbersCA086438, CA083084, CA008748, RR024996, and CA161280 awarded by NationalInstitutes of Health. The government has certain rights in theinvention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Nov. 7, 2017, isnamed 2003080-1330_SL.txt and is 2,007 bytes in size.

FIELD OF THE INVENTION

The present invention relates to fluorescent silica-based nanoparticles,and methods of using the nanoparticles to detect, diagnose, or treatdiseases such as cancer.

BACKGROUND OF THE INVENTION

Early tumor detection and treatment selection is paramount to achievingtherapeutic success and long-term survival rates. At its early stage,many cancers are localized and can be treated surgically. However, insurgical settings, the evaluation of metastatic disease spread and tumormargins, particularly in areas of complex anatomy, is limited by a lackof imaging technologies. This has led to a disproportionate number ofinvasive biopsies. Molecularly-targeted probes incorporatingcontrast-producing (i.e., optical, PET) labels and offering improvedspecificity are needed for early imaging detection of moleculardifferences between normal and tumor cells, such as cancer-specificalterations in receptor expression levels. When combined withhigher-sensitivity and higher-resolution imaging tools, specificmolecular-targeted probes will greatly improve detection sensitivity,staging, and the monitoring and/or treatment of cancer.

Current fluorescence imaging probes typically consist of singleconventional fluorophore (e.g., organic dyes, fluorescent proteins),fluorescent proteins (e.g., GFP) and semiconductor quantum dots(Q-dots). Single fluorophores are usually not stable and have limitedbrightness for imaging. Similar to dyes, the fluorescent proteins tendto exhibit excited state interactions which can lead to stochasticblinking, quenching and photobleaching. Q-dots are generally made fromheavy metal ions such as Pb²⁺ or Cd²⁺ and, therefore, are toxic. Burnset al. “Fluorescent core-shell silica nanoparticles: towards “Lab on aParticle” architectures for nanobiotechnology”, Chem. Soc. Rev., 2006,35, 1028-1042.

Fluorescent nanoparticles having an electrically conducting shell and asilica core are known and have utility in modulated delivery of atherapeutic agent. U.S. Pat. Nos. 6,344,272, and 6,428,811. Ashortcoming of existing fluorescent nanoparticles is their limitedbrightness and their low detectability as fluorescent probes indispersed systems.

The present multifunctional fluorescent silica-based nanoparticles offermany advantages over other typically larger diameter particle probes.The nanoparticles are non-toxic, exhibit excellent photophysicalproperties (including fluorescent efficiency and photostability), anddemonstrate enhanced binding affinity, potency, as well as a distinctpharmacokinetic signature—one in which bulk renal clearance predominateswithout significant reticuloendothelial system (RES) uptake. Theirrelatively small size, and surface PEG coating facilitates excellentrenal clearance. The fluorescent nanoparticles of the present inventioncontain a fluorescent core and silica shell. The core-shellarchitectures, the great surface area and diverse surface chemistry ofthe nanoparticle permit multiple functionalities simultaneouslydelivered to a target cell. For example, the nanoparticle can befunctionalized with targeting moieties, contrast agents for medicalimaging, therapeutic agents, or other agents. The targeting moieties onthe surface of the nanoparticle may be tumor ligands, which, whencombined with nanoparticle-conjugated therapeutic agents, makes thenanoparticle an ideal vehicle for targeting and potentially treatingcancer. Webster et al. Optical calcium sensors: development of a genericmethod for their introduction to the cell using conjugated cellpenetrating peptides. Analyst, 2005; 130:163-70. The silica-basednanoparticle may be labeled with contrast agents for PET, SPECT, CT,MRI, and optical imaging.

SUMMARY

The present application provides for a method for detecting tumor cellscomprising the steps of: (a) administering to a patient a plurality offluorescent silica-based nanoparticles in a dose ranging from about 0.01nanomole/kg body weight to about 1 nanomole/kg body weight, thenanoparticle comprising: a silica-based core comprising a fluorescentcompound positioned within the silica-based core; a silica shellsurrounding at least a portion of the core; an organic polymer attachedto the nanoparticle; a ligand attached to the nanoparticle and capableof binding a tumor marker; and at least one therapeutic agent; and (b)detecting the nanoparticles.

The nanoparticle may be administered subdermally, peritumorally, orally,intravenously, nasally, subcutaneously, intramuscularly ortransdermally.

A fluorescent silica-based nanoparticle comprising:

The present invention also provides for a fluorescent silica-basednanoparticle comprising: a silica-based core comprising a fluorescentcompound positioned within the silica-based core; a silica shellsurrounding at least a portion of the core; an organic polymer attachedto the nanoparticle; and a ligand attached to the nanoparticle, whereinthe nanoparticle has a diameter between about 1 nm and about 15 nm, andafter administration of the nanoparticle to a subject, renal clearanceof the nanoparticle ranges from about 80% ID (initial dose) to about100% ID in about 24 hours, or from about 90% ID to about 100% ID inabout 24 hours.

The present invention provides a fluorescent silica-based nanoparticlecomprising a silica-based core having a fluorescent compound positionedwithin the silica-based core; a silica shell surrounding at least aportion of the core; an organic polymer attached to the nanoparticle;from about 1 to about 30 ligands, or from about 1 to about 20 ligandsattached to the nanoparticle; and a contrast agent or a chelate attachedto the nanoparticle.

The diameter of the nanoparticle ranges from about 1 nm to about 25 nm,or from about 1 nm to about 8 nm. The organic polymers that may beattached to the nanoparticle include poly(ethylene glycol) (PEG),polylactate, polylactic acids, sugars, lipids, polyglutamic acid (PGA),polyglycolic acid, poly(lactic-co-glycolic acid) (PLGA), Polyvinylacetate (PVA), or the combinations thereof.

The ligand may be capable of binding to at least one cellular component,such as a tumor marker. The number of ligands attached to thenanoparticle may also range from about 1 to about 30, from about 1 toabout 25, or from about 1 to about 10. Examples of the ligand includepeptide, protein, biopolymer, synthetic polymer, antigen, antibody,microorganism, virus, receptor, hapten, enzyme, hormone, chemicalcompound, pathogen, toxin, surface modifier, or combinations thereof.Peptides such as tripeptide RGD, cyclic peptide cRGD, cyclic peptidecRGDYC (SEQ ID NO: 7), octreotate, EPPT1 and peptide analogs ofalpha-MSH are encompassed by the present invention. Any linear, cyclicor branched peptide containing the RGD or alpha-MSH sequence is withinthe scope of the present invention.

A contrast agent, such as a radionuclide including ⁸⁹Zr, ⁶⁴Cu, ⁶⁸Ga,⁸⁶Y, ¹²⁴I and ¹⁷⁷Lu, may be attached to the nanoparticle. Thenanoparticle may be attached to a chelate, for example, DFO, DOTA, TETAand DTPA, that is adapted to bind a radionuclide.

The nanoparticle of the present invention may be detected by positronemission tomography (PET), single photon emission computed tomography(SPECT), computerized tomography (CT), magnetic resonance imaging (MRI),optical imaging (such as fluorescence imaging including near-infraredfluorescence (NIRF) imaging), bioluminescence imaging, or combinationsthereof.

A therapeutic agent may be attached to the nanoparticle. The therapeuticagents include antibiotics, antimicrobials, antiproliferatives,antineoplastics, antioxidants, endothelial cell growth factors, thrombininhibitors, immunosuppressants, anti-platelet aggregation agents,collagen synthesis inhibitors, therapeutic antibodies, nitric oxidedonors, antisense oligonucleotides, wound healing agents, therapeuticgene transfer constructs, extracellular matrix components,vasodialators, thrombolytics, antimetabolites, growth factor agonists,antimitotics, statin, steroids, steroidal and non-steroidalanti-inflammatory agents, angiotensin converting enzyme (ACE)inhibitors, free radical scavengers, PPAR-gamma agonists, smallinterfering RNA (siRNA), microRNA, and anti-cancer chemotherapeuticagents. The therapeutic agents encompassed by the present invention alsoinclude radionuclides, for example, ⁹⁰Y, ¹³¹I and ¹⁷⁷Lu. The therapeuticagent may be radiolabeled, such as labeled by binding to radiofluorine¹⁸F.

After administration of the nanoparticle to a subject, blood residencehalf-time of the nanoparticle may range from about 2 hours to about 25hours, from about 3 hours to about 15 hours, or from about 4 hours toabout 10 hours. Tumor residence half-time of the nanoparticle afteradministration of the nanoparticle to a subject may range from about 5hours to about 5 days, from about 10 hours to about 4 days, or fromabout 15 hours to about 3.5 days. The ratio of tumor residence half-timeto blood residence halftime of the nanoparticle after administration ofthe nanoparticle to a subject may range from about 2 to about 30, fromabout 3 to about 20, or from about 4 to about 15. Renal clearance of thenanoparticle after administration of the nanoparticle to a subject mayrange from about 10% ID (initial dose) to about 100% ID in about 24hours, from about 30% ID to about 80% ID in about 24 hours, or fromabout 40% ID to about 70% ID in about 24 hours. In one embodiment, afterthe nanoparticle is administered to a subject, blood residence half-timeof the nanoparticle ranges from about 2 hours to about 25 hours, tumorresidence half-time of the nanoparticle ranges from about 5 hours toabout 5 days, and renal clearance of the nanoparticle ranges from about30% ID to about 80% ID in about 24 hours.

When the nanoparticles in the amount of about 100 times of the humandose equivalent are administered to a subject, substantially no anemia,weight loss, agitation, increased respiration, GI disturbance, abnormalbehavior, neurological dysfunction, abnormalities in hematology,abnormalities in clinical chemistries, drug-related lesions in organpathology, mortality, or combinations thereof, is observed in thesubject in about 10 to about 14 days.

The present invention also provides a fluorescent silica-basednanoparticle comprising a silica-based core comprising a fluorescentcompound positioned within the silica-based core; a silica shellsurrounding at least a portion of the core; an organic polymer attachedto the nanoparticle; and a ligand attached to the nanoparticle, whereinthe nanoparticle has a diameter between about 1 nm and about 15 nm.After administration of the nanoparticle to a subject, blood residencehalf-time of the nanoparticle may range from about 2 hours to about 25hours, or from about 2 hours to about 15 hours; tumor residencehalf-time of the nanoparticle may range from about 5 hours to about 2days; and renal clearance of the nanoparticle may range from about 30%ID to about 80% ID in about 24 hours. The number of ligands attached tothe nanoparticle may range from about 1 to about 20, or from about 1 toabout 10. The diameter of the nanoparticle may be between about 1 nm andabout 8 nm. A contrast agent, such as a radionuclide, may be attached tothe nanoparticle. Alternatively, a chelate may be attached to thenanoparticle. The nanoparticle may be detected by PET, SPECT, CT, MRI,optical imaging, bioluminescence imaging, or combinations thereof. Atherapeutic agent may be attached to the nanoparticle. Afteradministration of the nanoparticle to a subject, blood residencehalf-time of the nanoparticle may also range from about 3 hours to about15 hours, or from about 4 hours to about 10 hours. Tumor residencehalf-time of the nanoparticle after administration of the nanoparticleto a subject may also range from about 10 hours to about 4 days, or fromabout 15 hours to about 3.5 days. The ratio of tumor residence half-timeto blood residence half-time of the nanoparticle after administration ofthe nanoparticle to a subject may range from about 2 to about 30, fromabout 3 to about 20, or from about 4 to about 15. Renal clearance of thenanoparticle may also range from about 45% ID to about 90% ID in about24 hours after administration of the nanoparticle to a subject.

Also provided in the present invention is a fluorescent silica-basednanoparticle comprising a silica-based core comprising a fluorescentcompound positioned within the silica-based core; a silica shellsurrounding at least a portion of the core; an organic polymer attachedto the nanoparticle; and a ligand attached to the nanoparticle, whereinthe nanoparticle has a diameter between about 1 nm and about 8 nm. Afteradministration of the nanoparticle to a subject, the ratio of tumorresidence half-time to blood residence half-time of the nanoparticleranges from about 2 to about 30, and renal clearance of the nanoparticleranges from about 30% ID to about 80% ID in about 24 hours.

The present invention further provides a method for detecting acomponent of a cell comprising the steps of: (a) contacting the cellwith a fluorescent silica-based nanoparticle comprising a silica-basedcore comprising a fluorescent compound positioned within thesilica-based core; a silica shell surrounding at least a portion of thecore; an organic polymer attached to the nanoparticle; from about 1 toabout 30 ligands attached to the nanoparticle; and a contrast agent or achelate attached to the nanoparticle; and (b) monitoring the binding ofthe nanoparticle to the cell or a cellular component by at least oneimaging technique.

The present invention further provides a method for targeting a tumorcell comprising administering to a cancer patient an effective amount ofa fluorescent silica based nanoparticle comprising a silica-based corecomprising a fluorescent compound positioned within the silica-basedcore; a silica shell surrounding at least a portion of the core; anorganic polymer attached to the nanoparticle; a ligand attached to thenanoparticle and capable of binding a tumor marker; and at least onetherapeutic agent. The nanoparticle may be radiolabeled. Thenanoparticle may be administered to the patient by, but not restrictedto, the following routes: oral, intravenous, nasal, subcutaneous, local,intramuscular or transdermal.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1A shows a dynamic light scattering (DLS) plot (number average) ofparticle size for bare silica (gray) and PEG-coated (black)Cy5-containing silica nanoparticles.

FIG. 1B shows in vivo imaging of spectrally demixed Cy5 particlefluorescence (pseudocolor) overlaid on visible light imaging of nudemice 45 min post-injection with bare silica nanoparticles.

FIG. 1C shows in vivo imaging of spectrally demixed Cy5 particlefluorescence (pseudocolor) overlaid on visible light imaging of nudemice 45 min post-injection with PEG-ylated Cy5 nanoparticles.

FIG. 1D shows in vivo biodistribution study using co-registered PET-CT.Upper row is serial co-registered PET-CT image 24-hr after injection of¹²⁴I-labeled PEG coated nanoparticle, flanked by the independentlyacquired microCT and microPET scans. Lower row is serial microPETimaging.

FIG. 2A shows fluorescence correlation spectroscopy (FCS) data andsingle exponential fits for Cy5 dye (light gray), 3.3±0.06 nm diameter(dark gray, mean±standard deviation, n=9) and 6.0±0.1 nm diameter(black, mean±standard deviation, n=6) Cy5-containing PEG-coatednanoparticles showing the differences in diffusion time resulting fromthe different hydrodynamic sizes of the different species.

FIG. 2B shows absorption and emission spectra of Cy5 dye (light gray),3.3 nm diameter (dark gray) and 6.0 nm diameter (black) PEG-coatednanoparticles.

FIG. 2C shows relative brightness comparison of free dye (light gray)with 3.3 nm (dark gray) and 6.0 nm diameter (black) nanoparticles,measured as count rate per molecule/particle as determined from the FCScurves.

FIG. 2D shows photobleaching data for Cy5 dye (light gray), 3.3 nmdiameter (dark gray), and 6.0 nm diameter (black) PEG-coatednanoparticles under ˜3.5 mW laser excitation.

FIG. 3A shows percent of initial particle dose (% ID) retained by blood(black) and tissues: liver (light gray), lung (mid-low gray), spleen(midgray), and kidney (mid-high gray) for 6.0 nm diameter nanoparticlesat various time points from 10 min to 48 h post-injection (n=3 mice,mean±standard deviation).

FIG. 3B shows plot of retained particle concentration for 3.3 nm (lightgray) and 6.0 nm (black) diameter nanoparticles and the associatedlogarithmic decay fits and half-lives.

FIG. 3C shows plot of estimated particle excretion for 3.3 nm (lightgray) and 6.0 nm (black) diameter nanoparticles and the associatedlogarithmic fits and half-lives (mean±standard deviation, n=9 (threemice per time point)).

FIGS. 4A-4C show in vivo biodistribution of the nanoparticles innon-tumor-bearing and tumor-bearing mice with subcutaneous C6xenografts. (A) Bare silica particles; (B) PEGylated RGD particles.

FIGS. 5A-5B show total specific binding data for cRGD- and PEG-ylateddots (i.e., nanoparticles) using flow cytometry in the Cy5 channel as afunction of time (FIG. 5A) and particle concentration (FIG. 5B).

FIGS. 6A-6B show multimodal C dot design for α_(v)β₃-integrin targetingand characterization.

FIG. 6A. Schematic representation of the ¹²⁴I-cRGDY-PEG-ylatedcore-shell silica nanoparticle with surface-bearing radiolabels andpeptides and core-containing reactive dye molecules (insets). “RGDY”disclosed as SEQ ID NO: 1.

FIG. 6B. FCS results and single exponential fits for measurements of Cy5dyes in solution (black), PEGcoated (PEG-dot, red), and PEG-coated,cRGDY (SEQ ID NO: 1)—labeled dots (blue, underneath red data set)showing diffusion time differences as a result of varying hydrodynamicsizes.

FIG. 6C. Hydrodynamic sizes (mean±s.d., n=15), and relative brightnesscomparisons of the free dye with PEG-coated dots and cRGDY-PEG dotsderived from the FCS curves, along with the corresponding dye andparticle concentrations.

FIG. 7 shows purification and quality control of ¹²⁴I-RGDY-PEG-dotsusing size exclusion column chromatography. Radioactivity (right column)of ¹²⁴I-RGDdots and ¹²⁴I-PEG-dots detected by γ-counting andcorresponding fluorescence signal intensity (Cy5, left column) of¹²⁴I-RGDY-PEG-dots and ¹²⁴I-PEG-dots in each eluted fraction.

FIGS. 8A-8D show competitive integrin receptor binding studies with¹²⁴I-cRGDY-PEG-dots, cRGDY peptide (SEQ ID NO: 1), and anti-α_(v)β₃antibody using two cell types. FIG. 8A. High affinity and specificbinding of ¹²⁴I-cRGDY-PEG-dots to M21 cells by γ-counting. Inset showsScatchard analysis of binding data plotting the ratio of theconcentration receptor-bound (B) to unbound (or free, F) radioligand, orbound-to-free ratio, B/F, versus the receptor-bound receptorconcentration, B; the slope corresponds to the dissociation constant,Kd.

FIG. 8B. α_(v)β₃-integrin receptor blocking of M21 cells using flowcytometry and excess unradiolabeled cRGD or anti-α_(v)β₃ antibody priorto incubation with cRGDY-PEG-dots.

FIG. 8C. Specific binding of cRGDY-PEG-dots to M21 as against M21L cellslacking surface integrin expression using flow cytometry.

FIG. 8D. Specific binding of cRGDY-PEG-dots to HUVEC cells by flowcytometry. Each bar represents mean±s.d. of three replicates.

FIGS. 9A-9D show pharmacokinetics and excretion profiles of the targetedand non-targeted particle probes.

FIG. 9A. Biodistribution of ¹²⁴I-cRGDY-PEG-dots in M21 tumor-bearingmice at various times from 4 to 168 h p.i. The inset shows arepresentative plot of these data for blood to determine the residencehalf-time (T_(1/2)).

FIG. 9B. Biodistribution of ¹²⁴I-PEG-dots from 4 to 96 h postinjection.

FIG. 9C. Clearance profile of urine samples collected up to 168 hr p.i.of unradiolabeled cRGDY-PEG-dots (n=3 mice, mean±s.d.).

FIG. 9D. Corresponding cumulative % ID/g for feces at intervals up to168 hr p.i. (n=4 mice). For biodistribution studies, bars represent themean±s.d.

FIGS. 10A-10B show acute toxicity testing results.

FIG. 10A. Representative H&E stained liver at 400× (upper frames) andstained kidneys at 200× (lower frames). Mice were treated with a singledose of either non-radiolabeled ¹²⁷I-RGDY-PEG-dots or ¹²⁷I-PEG-coateddots (control vehicle) via intravenous injection and organs collected 14days later.

FIG. 10B. Average daily weights for each treatment group of the toxicitystudy. Scale bar in FIG. 10A corresponds to 100 μm.

FIGS. 11A-11B show serial in vivo PET imaging of tumor-selectivetargeting.

FIG. 11A. Representative whole-body coronal microPET images at 4 hrsp.i. demonstrating M21 (left, arrow) and M21L (middle, arrow) tumoruptakes of 3.6 and 0.7% ID/g, respectively, and enhanced M21 tumorcontrast at 24 hrs (right).

FIG. 11B. In vivo uptake of ¹²⁴I-cRGDY-PEG-dots in α_(v)β₃integrin-overexpressing M21 (black, n=7 mice) and non-expressing M21L(light gray, n=5 mice) tumors and ¹²⁴I-PEG-dots in M21 tumors (darkgray, n=5).

FIG. 11C. M21 tumor-to-muscle ratios for ¹²⁴I-cRGDY-PEG-dots (black) and¹²⁴I-PEG-dots (gray).

FIG. 11D. Correlation of in vivo and ex-vivo M21 tumor uptakes of cRGDY(SEQ ID NO: 1) labeled and unlabeled probes. Each bar represents themean±s.d.

FIGS. 12A-12B show nodal mapping using multi-scale near-infrared opticalfluorescence imaging.

FIG. 12A. Whole body fluorescence imaging of the tumor site (T) anddraining inguinal (ILN) and axillary (ALN) nodes and communicatinglymphatics channels (bar, LC) 1-hr p.i. in a surgically-exposed livinganimal.

FIG. 12B. Corresponding co-registered white-light and high-resolutionfluorescence images (upper row) and fluorescence images only (lower row)revealing nodal infrastructure of local and distant nodes, includinghigh endothelial venules (HEV). The larger scale bar in (12B)corresponds to 500 μm.

FIG. 13A shows the experimental setup of using spontaneous miniswinemelanoma model for mapping lymph node basins and regional lymphaticsdraining the site of a known primary melanoma tumor.

FIG. 13B shows small field-of-view PET image 5 minutes after subdermalinjection of multimodal particles (¹²⁴I-RGD-PEG-dots) about the tumorsite.

FIG. 14A shows whole-body dynamic ¹⁸F-fluorodeoxyglucose (¹⁸F-FDG) PETscan demonstrating sagittal, coronal, and axial images through the siteof nodal disease in the neck.

FIG. 14B shows fused ¹⁸F-FDG PET-CT scans demonstrating sagittal,coronal, and axial images through the site of nodal disease in the neck.

FIG. 14C shows the whole body miniswine image.

FIGS. 15A-15C show the same image sets as in FIGS. 14A-14C, but at thelevel of the primary melanoma lesion, adjacent to the spine on the upperback.

FIG. 16A shows high resolution dynamic PET images following subdermal,4-quadrant injection of ¹²⁴I-RGD-PEG-dots about the tumor site over a 1hour time period.

FIG. 16B shows fused PET-CT images following subdermal, 4-quadrantinjection of ¹²⁴I-RGD-PEG-dots about the tumor site over a 1 hour timeperiod.

FIG. 16C shows Cy5 imaging (top image), the resected node (second to topimage), and H&E staining (lower two images).

FIG. 17 shows a scheme for a nanoparticle with a fluorescent dye withinthe core and a PEG surface-coating. The nanoparticle is decorated withtriple bonds for subsequent “click chemistry” with both DFO andTyr3-octreotate functionalized with azide groups.

FIG. 18 shows structures of PEG derivative. Standard chemical reactionsare used for the production of the functionalized PEG with triple bonds,which will then be covalently attached to the nanoparticle via thesilane group.

FIG. 19 shows structures of DFO derivatives.

FIG. 20A shows structures of Tyr3-octreotate.

FIG. 20B shows synthesis of the azide-containing acid for incorporationinto Tyr3-Octreotate.

FIG. 21A shows a scheme of the production of functionalized nanoparticlewith an NIR fluorescent dye within its core, a PEG surface-coating, DFOchelates and Tyr3-octreotate.

FIG. 21B shows a scheme of the production of a multimodality⁸⁹Zr-labeled nanoparticle (PET and fluorescence) decorated withTyr3-octreotate.

FIG. 21C shows the tetrazine-norbornene ligation.

FIG. 21D shows a scheme of the strategy for the creation of radiolabeledcore-shell nanoparticles using the tetrazine-norbornene ligation.

FIG. 21E shows a scheme of the strategy for the creation ofpeptide-targeted radiolabeled core-shell nanoparticles using thetetrazine-norbornene ligation. Both one-step (in which thepre-metallated chelator-norbornene complex is reacted with the particle)and two-step (in which the chelator is metallated after conjugation)pathways are shown.

FIG. 22 shows microscopic images demonstrating co-localization betweencRGF-PEG-nanoparticles and lysotracker red in the endocytotic pathway.

FIGS. 23A-23I Image-guided SLN (sentinel lymph node) Mapping:Pre-operative PET imaging. (FIGS. 23A,23B) Axial CT images reveal a leftpelvic soft tissue mass (FIG. 23A, arrow) and left flank SLN (b, arrow).(FIGS. 23C, 23D) Axial ¹⁸F-FDG PET images show localized activity withinthe tumor (c, arrow) and left flank SLN (FIG. 23D, arrow) following i.v.tracer injection. (FIG. 23E) Axial and (FIG. 23F) coronal¹²⁴I-cRGDY-PEG-C dot co-registered PET-CT images show site of localinjection about the pelvic lesion (FIG. 23E, arrow). (FIG. 23G)Corresponding axial and (h) coronal co-registered PET-CT images localizeactivity to the SLN (FIG. 23G, arrow). (i) Radioactivity levels of theprimary tumor, SLN (in vivo, ex vivo), and a site remote from theprimary tumor (i.e., background), using a handheld gamma probe.

FIGS. 24A-24Q Image-guided SLN mapping: Real-time intraoperative opticalimaging with correlative histology. Intraoperative SLN mapping wasperformed on the animal shown in FIGS. 23A-23I. (FIGS. 24A-24I)Two-channel NIR optical imaging of the exposed nodal basin. Localinjection of Cy5.5-incorporated particles displayed in dual-channelmodel (FIG. 24A) RGB color and (FIG. 24B) NIR fluorescent channels(white). (FIGS. 24C-24F) Draining lymphatics distal to the site ofinjection. Fluorescence signal within the main draining proximal (FIGS.24C,24D), mid (FIG. 24E), and distal (FIG. 24F) lymphatic channels(arrows) extending toward the SLN (‘N’). Smaller caliber channels arealso shown (arrowheads). Images of the SLN displayed in the (FIG. 24G)color and (FIG. 24H) NIR channels. (FIG. 24I) Image of the exposed SLN.(FIG. 24J-24M) Images of SLN in the color and NIR channels during (FIG.24J,24K) and following (FIG. 24L, FIG. 24M) excision, respectively.(FIG. 24N) Low power view of H&E stained SLN shows cluster of pigmentedcells (black box) (bar=1 mm). (FIG. 24O) Higher magnification of (FIG.24N) reveals rounded pigmented melanoma cells and melanophages (bar=50μm). (FIG. 24P) Low power view of HMB45-stained SLN confirms presence ofmetastases (black box, bar=500 μm). (FIG. 24Q) Higher magnification in(FIG. 24P) reveals clusters of HMB45+ expressing melanoma cells (bar=100μm).

FIGS. 25A-25K Discrimination of inflammation from metastatic disease:Comparison of ¹⁸F-FDG and ¹²⁴I-cRGDY-PEG C dot tracers. (FIGS. 25A-25D)Imaging of inflammatory changes using ¹⁸F-FDG-PET with tissuecorrelation. (FIG. 25A) Axial CT scan of the ¹⁸F-FDG PET study showscalcification within the left posterior neck (arrows). (FIG. 25B) Fusedaxial ¹⁸F-FDG PET-CT reveals hypermetabolic activity at this same site(arrows). Increased PET signal is also seen in metabolically activeosseous structures (asterisks). (FIG. 25C) Low- and (FIG. 25D)high-power views of H&E-stained calcified tissue demonstrate extensiveinfiltration of inflammatory cells. (FIGS. 25E-25K) Metastatic diseasedetection following injection of ¹²⁴I-cRGDY-PEG C dots about the tumorsite. (FIG. 25E) Preinjection axial CT scan of ¹²⁴I-cRGDY-PEG-C dotsshows calcified soft tissues within the posterior neck (arrows). (FIG.25F) Co-registered PET-CT shows no evident activity corresponding tocalcified areas (arrow), but demonstrates a hypermetabolic node on theright (arrowhead). (FIG. 25G) Axial CT at a more superior level showsnodes (arrowheads) bilaterally and a calcified focus (arrow). (FIG. 25H)Fused PET-CT demonstrates PET-avid nodes (N) and lymphatic drainage(curved arrow). Calcification shows no activity (arrow). (FIG. 25I) Low-and (FIG. 25J) high-power views confirm the presence of nodalmetastases. (FIG. 25J) Single frame from a three-dimensional (3D) PETimage reconstruction shows multiple bilateral metastatic nodes(arrowheads) and lymphatic channels (arrow). Bladder activity is seenwith no significant tracer accumulation in the liver. Scale bars: 500 μm(FIG. 25C, FIG. 25D); 100 μm (FIG. 25I, FIG. 25J).

FIGS. 26A-26C show 3D Integrated ¹⁸F-FDG and ¹²⁴I-cRGDY-PEG-C dotPET-CT. (FIG. 26A-26C) 3D Volume rendered images were generated from CTand PET imaging data shown in FIGS. 25A-25K. (FIG. 26A) PET-CT fusionimage (coronal view) shows no evident nodal metastases (asterisks).Increased activity within bony structures is identified. (FIG. 26B, FIG.26C) High-resolution PET-CT fusion images showing coronal (FIG. 26B) andsuperior views (FIG. 26C) of bilateral metastatic nodes (open arrows)and lymphatic channels (curved arrows) within the neck following localparticle tracer injection.

FIGS. 27A-27O. Assessment of treatment response after radiofrequencyablation (RFA) using ¹²⁴I-cRGDY-PEG-C dots. (FIG. 27A-27C) Single-doseparticle radiotracer localization of the SLN. (FIG. 27A) Baselinecoronal CT (white arrowhead), (FIG. 27B) PET (black arrowhead), and(FIG. 27C) fused PET-CT images (white arrowhead) following a peritumoralinjection. (FIGS. 27B-27D) Tumor particle tracer activity. (FIG. 27B)PET-avid exophytic left pelvic mass (black arrow). (FIG. 27C, FIG. 27D)Combined PET-CT images showing a hypermetabolic lesion (white arrow) andparticle tracer flow within a draining lymphatic channel (asterisk)towards the SLN (curved arrow). (FIG. 27E, FIG. 27F) Pre-ablation axialCT images locate the SLN (e, white arrowhead) prior to RFA electrodeplacement (f, arrow) into the node (below crosshairs). (FIG. 27G)Pre-ablation fused PET-CT reveals increased SLN activity (posterior tocrosshairs). (FIG. 27H) Post-ablation PET-CT scan shows mildly reducedactivity at the SLN site, anterior to the needle tip. (FIG. 27I)Corresponding pre-ablation H&E staining of core biopsy tissue from theSLN confirms pigmented tumor infiltration (bar=200 μm). (FIG. 27J) Highmagnification of boxed area in (i) reveals large, rounded pigmentedclusters of melanoma cells (bar=50 μm). (FIG. 27K) Post-ablation H&Estaining shows necrotic changes within a partially tumor-infiltratednode (box) and multifocal hemorrhages (bar=500 μm). (FIG. 27L) Highmagnification of (FIG. 27K) reveals significant tissue necrosis(arrowheads) within the metastatic node, in addition to lymphoid tissue(bar=50 μm). (FIG. 27M) TUNEL staining of metastatic SLN before ablation(bar=20 μm). (FIG. 27N) Post-ablation TUNEL staining demonstrating focalareas of necrosis with adjacent scattered tumor foci and normal nodaltissue (NT) (bar=500 μm). (FIG. 27O) High magnification of boxed area in(FIG. 27N) shows positive TUNEL staining, consistent with necrosis(bar=20 μm).

FIGS. 28A-28C. Core-shell hybrid silica nanoparticle platform(¹²⁴I-cRGDY-PEG-C dots) and overview of study design. (FIG. 28A)Schematic of the hybrid (PET-optical) inorganic imaging probe (right)showing the core-containing deep-red dye and surface-attachedpolyethylene glycol (PEG) chains that bear cRGDY peptide (SEQ ID NO: 1)ligands and radiolabels for detecting human α_(v) β₃ integrin-expressingtumors (left). (FIG. 28B) Absorption-matched spectra (left: red, blackcurves) and emission spectra (right) for free (blue curve) andencapsulated (green curve) dyes revealing increased fluorescence ofencapsulated fluorophores. (FIG. 28C) Timeline of clinical trial events.Biol specs, biological specimens (blood, urine).

FIGS. 28D-28E. Whole body distribution and pharmacokinetics of¹²⁴I-cRGDY-PEG-C dots. (FIG. 28D) Maximum intensity projection (MIP) PETimages at 2-(left), 24-(middle) and 72-(right) hours p.i. of¹²⁴I-cRGDY-PEG-C dots reveal activity in bladder (*), heart (yellowarrow), and bowel (white arrowhead). (FIG. 28E) Decay-corrected percentinjected dose per gram (% ID/g) of urine and plasma collected atapproximately 30 min, 4 h, 24 h and 72 h following injection of theparticles was determined by gamma-counting; individual plots weregenerated for each patient. ROIs were drawn on major organs for eachpatient's PET scans for each patient to derive standardized uptakevalues and % ID/g.

FIGS. 29A-29K Metabolic analyses of biological specimens. (FIG. 29A,FIG. 29B) Time-dependent activity concentrations (% ID/g×100) in plasmaand urine, respectively, decay-corrected to the time of injection. (C-H)RadioTLC (4:1 acetic acid:methanol as mobile phase) of plasma and urinespecimens (decay-corrected counts per minute, cpm). (FIGS. 29C-29E)Chromatograms of plasma show a single peak at 0.5—(FIG. 29C), 3—(FIG.29D) and 24—(FIG. 29E) hours p.i. (FIG. 29F-29H). Chromatograms of urinespecimens reveal two peaks at 0.5—(FIG. 29F), 3—(FIG. 29G), 24—(FIG.29H) hours p.i. Insets (FIG. 29G, FIG. 29H) show respective data scaledto a maximum of 50 cpm. (FIG. 29I-FIG. 29K) Chromatograms of standards:injectate (FIG. 29I), radio-iodinated (¹³¹I) peptide (FIG. 29J) and free¹³¹I (FIG. 29K). Vertical lines discriminate peaks corresponding to theparticle tracer (long dashes; R_(f)=0.04), ¹³¹I-cRGDY (short dashes;R_(f)=0.2) and ¹³¹I (dotted; R_(f)=0.7).

FIGS. 30A-30C Whole-body PET-CT imaging of particle biodistribution andpreferential tumor uptake following systemic injection of¹²⁴I-cRGDY-PEG-C dots (FIG. 30A) Reformatted coronal CT demonstrates awell-defined, hypodense left hepatic lobe metastasis (arrowhead). (FIG.30B) Coronal PET image at 4 hours p.i. demonstrates increased activityalong the peripheral aspect of the tumor (arrowhead), in addition to thebladder, gastrointestinal tract (stomach, intestines), gallbladder, andheart. (FIG. 30C) Co-registered PET-CT localizes activity to the tumormargin.

FIGS. 31A-31J Multimodal imaging of particle uptake in a pituitarylesion. (FIGS. 31A-31B) Multiplanar contrast-enhanced MR axial (FIG.31A) and sagittal (FIG. 31B) images at 72 hours p.i. demonstrate asubcentimeter cystic focus (arrows) within the right aspect of theanterior pituitary gland. (FIGS. 31C-31D) Co-registered axial (FIG. 31C)and sagittal (FIG. 31D) MRI-PET images reveal increased focal activity(red) localized to the lesion site. (FIGS. 31E-31F) Axial (FIG. 31E) andsagittal (FIG. 31F) PET-CT images localize activity to the right aspectof the sella. (FIGS. 31G-31I) Axial PET images at 3 hours (FIG. 31G), 24hours (FIG. 31H) and 72 hours (FIG. 31I) p.i. demonstrate progressiveaccumulation of activity (SUVmax) within the sellar region along with acorresponding decline in background activity about the lesion. (FIG.31J) Tumor-to-brain (TB) and tumor-to-liver (T/L) activity ratiosincreasing as a function of post-injection times.

FIG. 32. Structure of N-Ac-Cys-(Ahx)₂-D-Lys-ReCCMSH (or alpha MSH)peptide used for nanoparticle conjugation.

FIG. 33. Structure of the original ReCCMSH targeting molecule.

FIGS. 34A and 34B. Competitive binding studies using a melanocortin-1receptor agonist (¹²⁵I-NDP). N-Ac-Cys-(Ahx)₂-D-Lys-ReCCMSH (oralpha-MSH) conjugated particles (FIG. 34A) had stronger affinity forcultured B16/F1 melanoma cells than a scrambled sequence version of themolecule (FIG. 34B).

FIGS. 35A and 35B. Dose-response data was obtained as a function oftargeted particle concentrations (FIG. 35A) and incubation times (FIG.35B) for both B16F10 and M21 melanoma cell lines.

FIG. 36. Human M21 cell survival studies, performed over a range ofparticle concentrations for a fixed incubation time of 48 hrdemonstrated no significant loss of cell viability.

FIGS. 37A and 37B. ¹²⁵I-radiolabeled alpha-MSH conjugated nanoparticlesdemonstrated bulk renal excretion over a 24 hr period in both B16F10 andM21 murine xenograft models.

FIGS. 38A and 38B. Neither B16F10 or M21 xenograft models showedsignificant accumulation of the targeted particle probe in thereticuloendothelial system (i.e., not an RES agent), nor in the kidney.

FIGS. 39A-39D. Competitive integrin receptor binding andtemperature-dependent uptake using cRGDY-PEG-C dots and anti-α_(v)β₃antibody for 2 cell types. FIG. 39A. Specific binding and uptake ofcRGDY-PEG-C dots in M21 cells as a function of temperature (4° C., 25°C., 37° C.) and concentration (25 nM, 100 nM) using anti-α_(v)β₃integrin receptor antibody and flow cytometry. Anti-α_(v)β₃ integrinreceptor antibody concentrations were 250 times (i.e., 250×) theparticle concentration. FIG. 39B. Uptake of cRGDY-PEG-C dots in M21cells, as against M21L cells lacking normal surface integrin expressionby flow cytometry. FIG. 39C. Selective particle uptake in HUVECs usinganti-α_(v)β₃ integrin receptor antibody and flow cytometry. FIG. 39D.cRGDY-PEG-C dot (1 red) colocalization assay with endocytic(transferrin-Alexa-488, FITC-dextran, green) and lysosomal markers(LysoTracker Red) after 4 h particle incubation using M21 cells.Colocalized vesicles (yellow), Hoechst counterstain (blue). Scale bar=15μm. Each data point (FIGS. 39A-39C) represents the mean±SD of 3replicates.

FIGS. 40A-40C. Expression levels of phosphorylated FAK, Src, MEK,Erk1/2, and Akt in M21 cells. FIG. 40A. FAK/Src complex transducesignals from integrin cell surface receptors via activation ofdownstream signaling pathways (PI3K-Akt, Ras-MAPK) to elicit a range ofbiological responses (boxes indicate assayed protein intermediates).FIG. 40B. Western blots of phosphorylated and total protein expressionlevels of key pathway intermediates after exposure (2 h, 37° C.) ofG₀/G₁ phase-synchronized M21 cells to 100 nM cRGDY-PEG-C-dots relativeto cells in serum-deprived (0.2% FBS) media (i.e., control). Aftertrypsinization of cells and re-suspension of the pellet in lysis buffer,proteins were resolved by 4-12% gradient SDS-PAGE and analyzed byanti-FAK 397, pFAK 576/577, p-Src, pMEK, pErk1/2, and pAkt antibodies.Antibodies against FAK, Src, MEK, Erk, and Akt were also used to detectthe amount of total protein. FIG. 40C. Graphical summary of percentsignal intensity changes in phosphorylated to total protein (AdobePhotoshop CS2; see Methods) for particle-exposed versus serum-deprivedcells. GF, growth factors; EC, endothelial cell; ECM, extracellularmatrix.

FIGS. 41A and 41B. Signaling induction and inhibition studies in M21cells using PF-573228 (PF-228), a FAK inhibitor. FIG. 41A. Western blotsof phosphorylated and total protein expression levels using theforegoing process of FIGS. 2A-2D with the addition of 250 nM or 500 nMPF-228 (0.5 h, 37° C.) to cells prior to particle exposure. FIG. 41B.Summary of percent intensity changes of phosphorylated to total proteinexpression levels in particle-exposed versus control cells with andwithout PF-228.

FIGS. 42A and 42B. Effect of cRGDY-PEG-C dots on M21 cellular migrationusing time lapse imaging. FIG. 42A. Time-dependent changes in cellmigration using ORIS™ collagen coated plates for a range of particleconcentrations (0-400 nM; 37° C.) in RPMI 1640 media supplemented with0.2% FBS, as against supplemented media alone (controls). Images werecaptured at time t=0 (pre-migration) and at subsequent 24 h intervalsfollowing stopper removal by a Zeiss Axiovert 200M inverted microscope(5×/0.25 NA objective) and a scan slide module (Metamorph® MicroscopyAutomation & Image Analysis Software) for a total of 96 hrs. FIG. 42B.Graphical plot of changes in the mean area of closure (%) as a functionof concentration using ImageJ software. Mean area of closure representsthe difference in the areas demarcated by the border of advancing cells(pixels) at arbitrary time points and after stopper removal (t=0),divided by the latter area. Quadruplicate samples were statisticallytested for each group using a one-tailed t-test: *, p=0.011; **,p=0.049; ***, p=0.036. Scale bars=100 μm and 33 μm (magnified images ofx and xv).

FIGS. 43A and 43B. Effect of cRGDY-PEG-C dots on the migration of HUVECcells. FIG. 43A. Serial HUVEC migration was assayed using the sameprocess in FIGS. 4A-4C over a 24-hr time interval. The displayed imagesand area of closure values indicated are representative of a singleexperiment. FIG. 43B. Mean areas of closure (%) were determined overthis time interval for a range of particle concentrations (0-400 nM)using ImageJ software. Triplicate assays were performed for eachconcentration and time point. One-tailed t-test *, p<0.05. Scale bar=76μm.

FIGS. 44A-44C. Inhibition of HUVEC cell migration using PF-228. FIG.44A. Same process as in FIGS. 5A-5B, except cells were exposed to 250 nMand 500 nM PF-228 (0.5 h, 37° C.) prior to particle exposure orincubation in 0.2% FCS supplemented media. FIG. 44B. Mean area closure(%) for cells incubated under the foregoing conditions. FIG. 44C.Tabulated p values for each exposure condition using a one-tailedt-test. Quadruplicate samples were run for each inhibitor concentration.Scale bar=50 μm.

FIGS. 45A-45C. Modulation of M21 cell spreading and adhesion usingcRGDY-PEG-C dots. FIG. 45A. Time-lapse imaging showing changes incellular attachment and spreading. Cells were pre-incubated in 0.2%FBS-supplemented RPMI (0.5 h, 25° C.), without and with particles (400nM), followed by seeding in (5 μg/ml) fibronectin-coated 96-well plates.Images were captured at t=0, 0.5 h, 1 h, and 2 h using a Zeiss Axiovert200M inverted microscope (20×/0.4 NA objective) and a scan slide modulein Metamorph®. FIG. 45B. Graphical plot showing the mean number ofrounded and elongated cells within two groups as a function of time:non-particle exposed (elongated, graph #1) and particle exposed(rounded, graph #2). Cells in each of three wells of a 96-well platewere manually counted in a minimum of three high power fields (×200magnification) and averaged. FIG. 45C. Absorbance (λ=650 nm; SpectroMaxM5 microplate reader) values for 4% paraformaldehyde fixed cells,exposed to media or 400 nM cRGDY-PEG C-dots, and treated with methyleneblue reagent (1 ml; 1 h, 37° C.), as a measure of cellular attachment.Scale bar=30 μm. Quadruplicate samples were run for each group.

FIGS. 46A and 46B. Influence of cRGDY-PEG-C dots on cell cycle. FIG.46A. Percentage (%) of viable cells in the G₁, S, and G₂ phases of thecell cycle as a function of particle concentration (0, 100, 300 nM)added to G₀/G₁-phase-synchronized M21 cells incubated for a total of 96hours. Italicized numbers above each bar represent the percentage ofcells (particle-incubated, control) in S, G₁ and G₂ phase, determined byflow cytometry. FIG. 46B. Representative cell cycle histograms are shownfor cells under control (i.e., no particle) conditions and afterincubation with 100 and 300 nM particles. One-way ANOVA: *, p<0.05; **,p<0.005 relative to S-phase control. Insets: Group mean values (n=3)±SDfor each cell cycle phase. Experiments were performed in triplicate.

FIGS. 47A and 47B. Dose-response effects and saturation binding kineticsusing cRGDY-PEG-C dots and α_(v)β₃ integrin-expressing cells. FIGS. 47A,47B. Cellular binding/uptake as a function of particle concentration(FIG. 47A) and incubation time (FIG. 47B) by flow cytometry. Monolayersof M21 and HUVEC cells were incubated (4 h, 25° C.) with increasingconcentrations of particles (5-600 nM), as well as over a range ofincubation times (0.5-4 h; 100 nM) and assayed by fluorescence-activatedcell sorting (FACS) analysis. The percentage of the total eventsdetected is displayed. Each data point represents mean±SD of 3replicates.

FIGS. 48A and 48B. Competitive integrin receptor binding with¹²⁴I-cRGDY-PEG-C dots and cRGDY peptide (SEQ ID NO: 1) in two celltypes. FIG. 48A. Specific binding of ¹²⁴I-cRGDY-PEG-dots to M21 andHUVEC cells following incubation with excess cRGDY peptide (SEQ IDNO: 1) using gamma counting. Monolayers of M21 and HUVEC cells wereincubated (4 h, 25° C.) with 25 nM of ¹²⁴I-cRGDY-PEG-dots in thepresence and absence of cRGDY peptide (SEQ ID NO: 1) (85, 170 nM).Binding is expressed as a percentage of the control (i.e.,radioiodinated cRGDY-PEG-dots). FIG. 48B. Tumor-directed binding ofnon-radiolabeled cRGDY-PEG-C dots. M21 and HUVEC cells were incubatedfor 4 h at 25° C. with two targeted particle concentrations (25 nM, 100nM) or particle controls (PEG-C dots) and assayed by flow cytometry.

FIGS. 49A-49D. Viability and proliferation of M21 and HUVEC cells as afunction of particle concentration and incubation time. FIGS. 49A, 49B.Absorbance (λ_(ex)=440 nm) as a measure of viability in subconfluent,G₀/G₁-phase-synchronized M21 (FIG. 49A) and HUVEC (FIG. 49B) cells overa 24 hour period using media supplemented with 10% or 2% FBS alone,respectively, or with addition of particles (25-200 nM). FIGS. 49C, 49D.Cellular proliferative activity in M21 (FIG. 49C) and HUVEC (FIG. 49D)cells over a 93 h time interval using either media alone orparticle-containing media, as specified in FIGS. 49A, 49B.

FIG. 50. Cell signaling changes as a function of particle incubationtime in M21 cells. Western blots of selected phosphorylated and totalprotein intermediates over an 8 h time period. Normalized intensityratios (i.e., difference of phospho-protein and total protein divided bythe latter) are graphically illustrated.

FIGS. 51A and 51B. Cell signaling modulation as a function of particleconcentration in M21 cells. Western blots of selected phosphorylated andtotal protein intermediates over a range of particle concentrations(i.e., 0-400 nM).

FIGS. 52A and 52B. Cell signaling inhibition studies in M21 cells. FIG.52A. Western blots of selected phosphorylated and total proteinintermediates (serum-deprived media, 100 nM particles alone, or 100 nMparticles after addition of 250 nM or 500 nM inhibitor). FIG. 52B.Relative intensities of phospho-protein and β-actin blots in FIG. 52Arelative to control cells (0.2% FBS).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a fluorescent silica-based nanoparticlethat allows for precise detection, characterization, monitoring andtreatment of a disease such as cancer. The invention also provides for amethod for detecting tumor cells. The method may contain the followingsteps: (a) administering to a patient a plurality of fluorescentsilica-based nanoparticles in a dose ranging from about 0.01 nanomole/kgbody weight to about 1 nanomole/kg body weight, from about 0.05nanomole/kg body weight to about 0.9 nanomole/kg body weight, from about0.1 nanomole/kg body weight to about 0.9 nanomole/kg body weight, fromabout 0.2 nanomole/kg body weight to about 0.8 nanomole/kg body weight,from about 0.3 nanomole/kg body weight to about 0.7 nanomole/kg bodyweight, from about 0.4 nanomole/kg body weight to about 0.6 nanomole/kgbody weight, or from about 0.2 nanomole/kg body weight to about 0.5nanomole/kg body weight, and (b) detecting the nanoparticles. In oneembodiment, the nanoparticle comprises a silica-based core having afluorescent compound positioned within the silica-based core; a silicashell surrounding at least a portion of the core; an organic polymerattached to the nanoparticle; a ligand attached to the nanoparticle andcapable of binding a tumor marker; and at least one therapeutic agent.

The nanoparticle has a range of diameters including between about 0.1 nmand about 100 nm, between about 0.5 nm and about 50 nm, between about 1nm and about 25 nm, between about 1 nm and about 15 nm, or between about1 nm and about 8 nm. The nanoparticle has a fluorescent compoundpositioned within the nanoparticle, and has greater brightness andfluorescent quantum yield than the free fluorescent compound. Thenanoparticle also exhibits high biostability and biocompatibility. Tofacilitate efficient urinary excretion of the nanoparticle, it may becoated with an organic polymer, such as poly(ethylene glycol) (PEG). Thesmall size of the nanoparticle, the silica base and the organic polymercoating minimizes the toxicity of the nanoparticle when administered invivo. In order to target a specific cell type, the nanoparticle mayfurther be conjugated to a ligand, which is capable of binding to acellular component (e.g., the cell membrane or other intracellularcomponent) associated with the specific cell type, such as a tumormarker or a signaling pathway intermediate. In one embodiment, atherapeutic agent may be attached to the nanoparticle. To permit thenanoparticle to be detectable by not only optical imaging (such asfluorescence imaging), but also other imaging techniques, such aspositron emission tomography (PET), single photon emission computedtomography (SPECT), computerized tomography (CT), and magnetic resonanceimaging (MM), the nanoparticle may also be conjugated to a contrastagent, such as a radionuclide.

The properties of the nanoparticles lead to bulk excretion through thekidneys, increased potency relative to a native peptide ligand, enhanceduptake, and preferential accumulation in tumors compared with normaltissues. This, along with the lack of in vivo toxicity, has resulted ina unique product resulting in its translation to the clinic.

The present particles can be used to preferentially detect and localizetumors. For example, nanomolar particle tracer doses administered in amicrodosing regime accumulate and preferentially localize at sites ofdisease, although not optimized for targeted detection.

The present nanoparticles may exhibit distinct and reproducible humanpharmacokinetic signatures in which renal clearance predominates (e.g.,renal clearance of the nanoparticle is greater than about 90% ID inabout 24 hours after administration of the nanoparticle to a subject)without significant RES uptake (e.g., less than about 10%).

The present nanoparticles are excellent diagnostic probes, exhibitingoptimal physicochemical properties in humans that enable them to “targetand clear” the body over relatively short time intervals (e.g., hours,days, etc.).

The present particles bearing multiple actively targeted ligands (e.g.,cRGDY (SEQ ID NO: 1) and alpha-MSH) demonstrate enhanced bindingaffinity to the cellular targets compared to the affinity of a ligandalone. In some embodiments, the present particles binds to a cellulartarget from about 2 to about 20 fold greater, from about 3 to about 15fold greater, from about 5 to about 10 fold greater than a ligand alone.

In certain embodiments, dual-modality, targeted particles specificallyassess tumor burden and can discriminate metastatic tumor from chronicinflammatory disease in large animal models of metastatic melanoma.

Targeted particles may enhance receptor binding affinity and avidity,increase plasma residence times, bioavailability and tumor retention,and/or promote intracellular delivery via internalization. Theutilization of such targeted probes within surgical (or medical)oncology settings may enable highly selective treatment ofcancer-bearing tissues, potentially reducing attendant complicationrates. The present particles may be advantageous over passively targetednanocarriers, as the latter penetrate and non-specifically accumulatewithin the tumor interstitium by enhanced permeability and retention(EPR) effects.

Particle-based imaging systems for cancer diagnostics should benon-toxic, and selectively detect sites of primary and metastaticdisease while exhibiting relatively rapid renal clearance. Under theseconditions, the likelihood of potential toxicity will be reduced giventhe smaller area under the plasma concentration-time curve. In oneembodiment, these renal clearance properties may be achieved byultrasmall particle-based platforms or macromolecular systems that meeteffective renal glomerular filtration size cutoffs of 10 nm or less.Absence of single-dose acute toxicity and the minimization of such riskswill also be important. A platform design should maximize safety throughrapid whole-body clearance and the selection of biokinetic profiles thatminimize non-specific uptake in the reticuloendothelial system (RES),thus reducing potential adverse exposures.

In one embodiment, the present particles are safe and stable in vivo.The particles exhibit distinctly unique and reproducible PK signaturesdefined by renal excretion. Coupled with preferential uptake andlocalization of the probe at sites of disease, these particles can beused in cancer diagnostics.

The nanoparticle may have both a ligand and a contrast agent. The ligandallows for the nanoparticle to target a specific cell type through thespecific binding between the ligand and the cellular component. Thistargeting, combined with multimodal imaging, has multiple uses. Forexample, the nanoparticles can be used to map metastatic disease, suchas mapping sentinel lymph nodes (SLN), as well as identifying tumormargins or neural structures, enabling the surgeon to resect malignantlesions under direct visualization and to obviate complications duringthe surgical procedure. The ligand may also facilitate entry of thenanoparticle into the cell or barrier transport, for example, forassaying the intracellular environment.

The nanoparticle can be coupled with a ligand and a therapeutic agentwith or without a radiolabel. The radiolabel can additionally serve as atherapeutic agent for creating a theranostic platform. This couplingallows the therapeutic particle to be delivered to the specific celltype through the specific binding between the ligand and the cellularcomponent. This specific binding of the therapeutic agent ensuresselective treatment of the disease site with minimum side effects.

Nanoparticle Structure

The fluorescent nanoparticle of the present invention includes asilica-based core comprising a fluorescent compound positioned withinthe core, and a silica shell on the core. The silica shell may surroundat least a portion of the core. Alternatively, the nanoparticle may haveonly the core and no shell. The core of the nanoparticle may contain thereaction product of a reactive fluorescent compound and a co-reactiveorgano-silane compound. In another embodiment, the core of thenanoparticle may contain the reaction product of a reactive fluorescentcompound and a co-reactive organo-silane compound, and silica. Thediameter of the core may be from about 0.05 nm to about 100 nm, fromabout 0.1 nm to about 50 nm, from about 0.5 nm to about 25 nm, fromabout 0.8 nm to about 15 nm, or from about 1 nm to about 8 nm. The shellof the nanoparticle can be the reaction product of a silica formingcompound. The shell of the nanoparticle may have a range of layers. Forexample, the silica shell may be from about 1 to about 20 layers, fromabout 1 to about 15 layers, from about 1 to about 10 layers, or fromabout 1 to about 5 layers. The thickness of the shell may range fromabout 0.01 nm to about 90 nm, from about 0.02 nm to about 40 nm, fromabout 0.05 nm to about 20 nm, from about 0.05 nm to about 10 nm, or fromabout 0.05 nm to about 5 nm.

The silica shell of the nanoparticle may cover only a portion ofnanoparticle or the entire particle. For example, the silica shell maycover about 1 to about 100 percent, from about 10 to about 80 percent,from about 20 to about 60 percent, or from about 30 to about 50 percentof the nanoparticle. The silica shell can be either solid, i.e.,substantially non-porous, meso-porous, such as semi-porous, or porous.

Synthesis of Nanoparticle

The present fluorescent nanoparticle may be synthesized by the steps of:covalently conjugating a fluorescent compound, such as a reactivefluorescent dye, with the reactive moeties including, but not limitedto, maleimide, iodoacetamide, thiosulfate, amine, N-Hydroxysuccimideester, 4-sulfo-2,3,5,6-tetrafluorophenyl (STP) ester, sulfosuccinimidylester, sulfodichlorophenol esters, sulfonyl chloride, hydroxyl,isothiocyanate, carboxyl, to an organo-silane compound, such as aco-reactive organo-silane compound, to form a fluorescent silicaprecursor, and reacting the fluorescent silica precursor to form afluorescent core; covalently conjugating a fluorescent compound, such asa reactive fluorescent dye, to an organo-silane compound, such as aco-reactive organo-silane compound, to form a fluorescent silicaprecursor, and reacting the fluorescent silica precursor with a silicaforming compound, such as tetraalkoxysilane, to form a fluorescent core;and reacting the resulting core with a silica forming compound, such asa tetraalkoxysilane, to form a silica shell on the core, to provide thefluorescent nanoparticle.

The synthesis of the fluorescent monodisperse core-shell nanoparticlesis based on a two-step process. First, the near-infrared organic dyemolecules (e.g., tetramethylrhodamine isothiocynate (TRITC)) arecovalently conjugated to a silica precursor and condensed to form adye-rich core. Second, the silica gel monomers are added to form adenser silica network around the fluorescent core material, providingshielding from solvent interactions that can be detrimental tophotostability. The versatility of the preparative route allows for theincorporation of different fluorescent compounds, such as fluorescentorganic compounds or dyes, depending on the intended nanoparticleapplication. The fluorescent compounds that may be incorporated in thedye-rich core can cover the entire UV-Vis to near-IR absorption andemission spectrum. U.S. patent application Ser. Nos. 10/306,614,10/536,569 and 11/119,969. Wiesner et al., PEG-coated Core-shell SilicaNanoparticles and Mathods of Manufactire and Use, PCT/US2008/74894.

For the synthesis of the compact core-shell nanoparticle, the dyeprecursor is added to a reaction vessel that contains appropriateamounts of ammonia, water and solvent and allowed to react overnight.The dye precursor is synthesized by addition reaction between a specificnear-infrared dye of interest and 3-aminopropyltriethoxysilane in molarratio of 1:50, in exclusion of moisture. After the synthesis of thedye-rich compact core is completed, tetraethylorthosilicate (TEOS) issubsequently added to grow the silica shell that surrounded the core.

The synthesis of the expanded core-shell nanoparticle is accomplished byco-condensing TEOS with the dye precursor and allowing the mixture toreact overnight. After the synthesis of the expanded core is completed,additional TEOS is added to grow the silica shell that surrounded thecore.

The synthesis of the homogenous nanoparticles is accomplished byco-condensing all the reagents, the dye precursor and TEOS and allowingthe mixture to react overnight.

Fluorescent Compound

The nanoparticles may incorporate any known fluorescent compound, suchas fluorescent organic compound, dyes, pigments, or combinationsthereof. A wide variety of suitable chemically reactive fluorescent dyesare known, see for example MOLECULAR PROBES HANDBOOK OF FLUORESCENTPROBES AND RESEARCH CHEMICALS, 6th ed., R. P. Haugland, ed. (1996). Atypical fluorophore is, for example, a fluorescent aromatic orheteroaromatic compound such as is a pyrene, an anthracene, anaphthalene, an acridine, a stilbene, an indole or benzindole, anoxazole or benzoxazole, a thiazole or benzothiazole, a4-amino-7-nitrobenz-2-oxa-1,3-diazole (NBD), a cyanine, a carbocyanine,a carbostyryl, a porphyrin, a salicylate, an anthranilate, an azulene, aperylene, a pyridine, a quinoline, a coumarin (includinghydroxycoumarins and aminocoumarins and fluorinated derivativesthereof), and like compounds, see for example U.S. Pat. Nos. 5,830,912,4,774,339, 5,187,288, 5,248,782, 5,274,113, 5,433,896, 4,810,636 and4,812,409. In one embodiment, Cy5, a near infrared fluorescent (NIRF)dye, is positioned within the silica core of the present nanoparticle.Near infrared-emitting probes exhibit decreased tissue attenuation andautofluorescence. Burns et al. “Fluorescent silica nanoparticles withefficient urinary excretion for nanomedicine”, Nano Letters, 2009, 9(1), 442-448.

Non-limiting fluorescent compound that may be used in the presentinvention include, Cy5, Cy5.5 (also known as Cy5++), Cy2, fluoresceinisothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC),phycoerythrin, Cy7, fluorescein (FAM), Cy3, Cy3.5 (also known as Cy3++),Texas Red, LightCycler-Red 640, LightCycler Red 705,tetramethylrhodamine (TMR), rhodamine, rhodamine derivative (ROX),hexachlorofluorescein (HEX), rhodamine 6G (R6G), the rhodaminederivative JA133, Alexa Fluorescent Dyes (such as Alexa Fluor 488, AlexaFluor 546, Alexa Fluor 633, Alexa Fluor 555, and Alexa Fluor 647),4′,6-diamidino-2-phenylindole (DAPI), Propidium iodide, AMCA, SpectrumGreen, Spectrum Orange, Spectrum Aqua, Lissamine, and fluorescenttransition metal complexes, such as europium. Fluorescent compound thatcan be used also include fluorescent proteins, such as GFP (greenfluorescent protein), enhanced GFP (EGFP), blue fluorescent protein andderivatives (BFP, EBFP, EBFP2, Azurite, mKalama1), cyan fluorescentprotein and derivatives (CFP, ECFP, Cerulean, CyPet) and yellowfluorescent protein and derivatives (YFP, Citrine, Venus, YPet).WO2008142571, WO2009056282, WO9922026.

The silica shell surface of the nanoparticles can be modified by usingknown cross-linking agents to introduce surface functional groups.Crosslinking agents include, but are not limited to, divinyl benzene,ethylene glycol dimethacrylate, trimethylol propane trimethacrylate,N,N′-methylene-bis-acrylamide, alkyl ethers, sugars, peptides, DNAfragments, or other known functionally equivalent agents. The ligand maybe conjugated to the nanoparticle of the present invention by, forexample, through coupling reactions using carbodiimide, carboxylates,esters, alcohols, carbamides, aldehydes, amines, sulfur oxides, nitrogenoxides, halides, or any other suitable compound known in the art. U.S.Pat. No. 6,268,222.

Organic Polymer

An organic polymer may be attached to the present nanoparticle, e.g.,attached to the surface of the nanoparticle. An organic polymer may beattached to the silica shell of the present nanoparticle. The organicpolymer that may be used in the present invention include PEG,polylactate, polylactic acids, sugars, lipids, polyglutamic acid (PGA),polyglycolic acid, poly(lactic-co-glycolic acid) (PLGA), polyvinylacetate (PVA), and the combinations thereof. The attachment of theorganic polymer to the nanoparticle may be accomplished by a covalentbond or non-covalent bond, such as by ionic bond, hydrogen bond,hydrophobic bond, coordination, adhesive, and physical absorption. Inone embodiment, the nanoparticle is covalently conjugated with PEG,which prevents adsorption of serum proteins, facilitates efficienturinary excretion and decreases aggregation of the nanoparticle. Burnset al. “Fluorescent silica nanoparticles with efficient urinaryexcretion for nanomedicine”, Nano Letters, 2009, 9 (1), 442-448.

The surface of the nanoparticle may be modified to incorporate at leastone functional group. The organic polymer (e.g., PEG) attached to thenanoparticle may be modified to incorporate at least one functionalgroup. For example, the functional group can be a maleimide orN-Hydroxysuccinimide (NETS) ester. The incorporation of the functionalgroup makes it possible to attach various ligands, contrast agentsand/or therapeutic agents to the nanoparticle.

Ligand

A ligand may be attached to the present nanoparticle. The ligand iscapable of binding to at least one cellular component. The cellularcomponent may be associated with specific cell types or have elevatedlevels in specific cell types, such as cancer cells or cells specific toparticular tissues and organs. Accordingly, the nanoparticle can targeta specific cell type, and/or provides a targeted delivery for thetreatment and diagnosis of a disease. As used herein, the term “ligand”refers to a molecule or entity that can be used to identify, detect,target, monitor, or modify a physical state or condition, such as adisease state or condition. For example, a ligand may be used to detectthe presence or absence of a particular receptor, expression level of aparticular receptor, or metabolic levels of a particular receptor. Theligand can be, for example, a peptide, a protein, a protein fragment, apeptide hormone, a sugar (i.e., lectins), a biopolymer, a syntheticpolymer, an antigen, an antibody, an antibody fragment (e.g., Fab,nanobodies), an aptamer, a virus or viral component, a receptor, ahapten, an enzyme, a hormone, a chemical compound, a pathogen, amicroorganism or a component thereof, a toxin, a surface modifier, suchas a surfactant to alter the surface properties or histocompatability ofthe nanoparticle or of an analyte when a nanoparticle associatestherewith, and combinations thereof. In one embodiment, the ligands areantibodies, such as monoclonal or polyclonal antibodies. In anotherembodiment, the ligands are receptor ligands. In still anotherembodiment, the ligand is poly-L-lysine (pLysine).

An antigen may be attached to the nanoparticle. The antigen-attachednanoparticle may be used for vaccination.

The terms “component of a cell” or “cellular component” refer to, forexample, a receptor, an antibody, a hapten, an enzyme, a hormone, abiopolymer, an antigen, a nucleic acid (DNA or RNA), a microorganism, avirus, a pathogen, a toxin, combinations thereof, and like components.The component of a cell may be positioned on the cell (e.g., atransmembrane receptor) or inside the cell. In one embodiment, thecomponent of a cell is a tumor marker. As used herein, the term “tumormarker” refers to a molecule, entity or substance that is expressed oroverexpressed in a cancer cell but not normal cell. For example, theoverexpression of certain receptors is associated with many types ofcancer. A ligand capable of binding to a tumor marker may be conjugatedto the surface of the present nanoparticle, so that the nanoparticle canspecifically target the tumor cell. A ligand may be attached to thepresent nanoparticle directly or through a linker.

The attachment of the ligand to the nanoparticle may be accomplished bya covalent bond or non-covalent bond, such as by ionic bond, hydrogenbond, hydrophobic bond, coordination, adhesive, and physical absorption.The ligand may be coated onto the surface of the nanoparticle. Theligand may be imbibed into the surface of the nanoparticle. The ligandmay be attached to the surface of the fluorescent nanoparticle, or maybe attached to the core when the shell is porous or is covering aportion of the core. When the ligand is attached to the nanoparticlethrough a linker, the linker can be any suitable molecules, such as afunctionalized PEG. The PEGs can have multiple functional groups forattachment to the nanoparticle and ligands. The particle can havedifferent types of functionalized PEGs bearing different functionalgroups that can be attached to multiple ligands. This can enhancemultivalency effects and/or contrast at the target site, which allowsthe design and optimization of a complex multimodal platform withimproved targeted detection, treatment, and sensing in vivo.

A variety of different ligands may be attached to the nanoparticle. Forexample, tripeptide Arg-Gly-Asp (RGD) may be attached to thenanoparticle. Alternatively, cyclic peptide cRGD (which may containother amino acid(s), e.g., cRGDY (SEQ ID NO: 1)) may be attached to thenanoparticle. Any linear, cyclic or branched peptide containing the RGDsequence is within the scope of the present invention. RGD binds toα_(v)β₃ integrin, which is overexpressed at the surface of activatedendothelial cells during angiogenesis and in various types of tumorcells. Expression levels of α_(v)β₃ integrin have been shown tocorrelate well with the aggressiveness of tumors. Ruoslahti et al. Newperspectives in cell adhesion: RGD and integrins. Science 1987; 238:491.Gladson et al. Glioblastoma expression of vitronectin and alpha v beta 3integrin. Adhesion mechanism for transformed glial cells. J. Clin.Invest. 1991; 88:1924-1932. Seftor et al. Role of the alpha v beta 3integrin in human melanoma cell invasion. Proc. Natl. Acad. Sci. 1992;89:1557-1561.

Synthetic peptide EPPT1 may be the ligand attached to the nanoparticle.EPPT1, derived from the monoclonal antibody (ASM2) binding site, targetsunderglycosylated MUC1 (uMUC1). MUC1, a transmembrane receptor, isheavily glycosylated in normal tissues; however, it is overexpressed andaberrantly underglycosylated in almost all human epithelial celladenocarcinomas, and is implicated in tumor pathogenesis. Moore et al.In vivo targeting of underglycosylated MUC-1 tumor antigen using amultimodal imaging probe. Cancer Res. 2004; 64:1821-7. Patel et al. MUC1plays a role in tumor maintenance in aggressive thyroid carcinomas.Surgery. 2005; 138:994-1001. Specific antibodies including monoclonalantibodies against uMUC1 may alternatively be conjugated to thenanoparticle in order to target uMUC1.

In one embodiment, peptide analogues of α-melanotropin stimulatinghormone (α-MSH) are the ligands attached to the nanoparticle. Peptideanalogues of α-MSH are capable of binding to melanocortin-1 receptors(MC1R), a family of G-protein-coupled receptors overexpressed inmelanoma cells. Loir et al. Cell Mol. Biol. (Noisy-le-grand) 1999,45:1083-1092.

In another embodiment, octreotate, a peptide analog of 14-amino acidsomatostatin, is the ligand attached to the nanoparticle. Octreotide,which has a longer half-life than somatostatin, is capable of binding tosomatostatin receptor (SSTR). SSTR, a member of the G-protein coupledreceptor family, is overexpressed on the surface of several humantumors. Reubi et al. Distribution of Somatostatin Receptors in Normaland Tumor-Tissue. Metab. Clin. Exp. 1990; 39:78-81. Reubi et al.Somatostatin receptors and their subtypes in human tumors and inperitumoral vessels. Metab. Clin. Exp. 1996; 45:39-41. Othersomatostatin analogs may alternatively be conjugated to the nanoparticleto target SSTR, such as Tyr3-octreotide (Y3-OC), octreotate (TATE),Tyr3-octreotate (Y3-TATE), and ¹¹¹In-DTPA-OC. These somatostatinanalogues may be utilized for both PET diagnostic imaging and targetedradiotherapy of cancer. de Jong et al. Internalization of radiolabelled[DTPA⁰]octreotide and [DOTA⁰, Tyr³]octreotide: peptides for somatostatinreceptor targeted scintigraphy and radionuclide therapy. Nucl. Med.Commun. 1998; 19:283-8. de Jong et al. Comparison of ¹¹¹In-LabeledSomatostatin Analogues for Tumor Scintigraphy and Radionuclide Therapy.Cancer Res. 1998; 58:437-41. Lewis et al. Comparison of four⁶⁴Cu-labeled somatostatin analogs in vitro and in a tumor-bearing ratmodel: evaluation of new derivatives for PET imaging and targetedradiotherapy. J Med Chem 1999; 42:1341-7. Krenning et al. SomatostatinReceptor Scintigraphy with Indium-111-DTPA-D-Phe-1-Octreotide in Man:Metabolism, Dosimetry and Comparison with Iodine-123-Tyr-3-Octreotide. JNucl. Med. 1992; 33:652-8.

Various ligands may be used to map sentinel lymph nodes (SLNs). SLNmapping may be used in diagnosing, staging and treating cancer. J591 isan anti-prostate-specific membrane antigen (i.e., anti-PSMA) monoclonalantibody. J591 has been previously used to detect and stage prostatecancer. Tagawa et al., Anti-prostate-specific membrane antigen-basedradioimmunotherapy for prostate cancer, Cancer, 2010, 116(4Suppl):1075-83. Bander et al., Targeting Metastatic Prostate Cancer withRadiolabeled Monoclonal Antibody J591 to the Extracellular Domain ofProstate Specific Membrane Antigen, The Journal of Urology 170(5),1717-1721 (2003). Wernicke et al., (2011) Prostate-Specific MembraneAntigen as a Potential Novel Vascular Target for Treatment ofGlioblastoma Multiforme, Arch Pathol Lab Med. 2011; 135:1486-1489. TheF(ab′)2 fragment of J591 may be used as a ligand attached to the presentnanoparticle. The nanoparticle may also be radiolabeled to create adual-modality probe. In one embodiment, nanoparticles bearing theF(ab′)2 fragment of J591 (e.g., HuJ591-F(ab′)2 fragments, or humanizedJ591-F(ab′)2 fragments) are used in diagnosing prostate cancer orendometrial cancer (e.g., endometrial endometrioid adenocarcinoma). Inanother embodiment, nanoparticles bearing the F(ab′)2 fragment of J591can be used to target brain tumor neovasculature for treatment, diseaseprogression monitoring. Brain tumor neovasculature has been found tooverexpress PSMA, as shown from prior immunohistochemistry evaluationsof excised high grade glioma specimens.

Cyclic peptides containing the sequence HWGF (SEQ ID NO: 2) are potentand selective inhibitors of MMP-2 and MMP-9 but not of several other MMPfamily members. Peptide CTTHWGFTLC (SEQ ID NO: 3) inhibits the migrationof human endothelial cells and tumor cells. Moreover, it prevents tumorgrowth and invasion in animal models and improves survival of micebearing human tumors. CTTHWGFTLC (SEQ ID NO: 3)—displaying phagespecifically targets angiogenic blood vessels in vivo. This peptide andits extension GRENYGHCTTHWGFTLC (SEQ ID NO: 4) or GRENYGHCTTHWGFTLS (SEQID NO: 5) can be used as ligands to be attached to the presentnanoparticle. The peptides can also be radiolabelled, e.g.,radioiodinated. Koivunen et al., Tumor targeting with a selectivegelatinase inhibitor, Nature Biotechnology 17, 768-774 (1999). PenateMedina et al., Liposomal tumor targeting in drug delivery utilizingMMP-2- and MMP-9-binding ligands, J. Drug Delivery, Volume 2011 (2011),Article ID 160515. Anticancer Research 21:4101-4106 (2005). In oneembodiment, ¹²⁴I labeled matrix metalloproteinase peptide inhibitor(MMPI)-attached nanoparticles are used for SLN mapping to stageendometrioid cancer.

The number of ligands attached to the nanoparticle may range from about1 to about 30, from about 1 to about 20, from about 2 to about 15, fromabout 3 to about 10, from about 1 to about 10, or from about 1 to about6. The small number of the ligands attached to the nanoparticle helpsmaintain the hydrodynamic diameter of the present nanoparticle whichmeets the renal clearance cutoff size range. Hilderbrand et al.,Near-infrared fluorescence: application to in vivo molecular imaging,Curr. Opin. Chem. Biol., 14:71-9, 2010. The number of ligands measuredmay be an average number of ligands attached to more than onenanoparticle. Alternatively, one nanoparticle may be measured todetermine the number of ligands attached. The number of ligands attachedto the nanoparticle can be measured by any suitable methods, which mayor may not be related to the properties of the ligands. For example, thenumber of cRGD peptides bound to the particle may be estimated usingFCS-based measurements of absolute particle concentrations and thestarting concentration of the reagents for cRGD peptide. Average numberof RGD peptides per nanoparticle and coupling efficiency of RGD tofunctionalized PEG groups can be assessed colorimetrically underalkaline conditions and Biuret spectrophotometric methods. The number ofligands attached to the nanoparticle may also be measured by othersuitable methods.

Contrast Agent

A contrast agent may be attached to the present nanoparticle for medicalor biological imaging. As used herein, the term “contrast agent” refersto a substance, molecule or compound used to enhance the visibility ofstructures or fluids in medical or biological imaging. The term“contrast agent” also refers to a contrast-producing molecule. Theimaging techniques encompassed by the present invention include positronemission tomography (PET), single photon emission computed tomography(SPECT), computerized tomography (CT), magnetic resonance imaging (MM),optical bioluminescence imaging, optical fluorescence imaging, andcombinations thereof. The contrast agent encompassed by the presentinvention may be any molecule, substance or compound known in the artfor PET, SPECT, CT, MM, and optical imaging. The contrast agent may beradionuclides, radiometals, positron emitters, beta emitters, gammaemitters, alpha emitters, paramagnetic metal ions, and supraparamagneticmetal ions. The contrast agents include, but are not limited to, iodine,fluorine, copper, zirconium, lutetium, astatine, yttrium, gallium,indium, technetium, gadolinium, dysprosium, iron, manganese, barium andbarium sulfate. The radionuclides that may be used as the contrast agentattached to the nanoparticle of the present invention include, but arenot limited to, ⁸⁹Zr, ⁶⁴Cu, ⁶⁸Ga, ⁸⁶Y, ¹²⁴I and ¹⁷⁷Lu.

The contrast agent may be directly conjugated to the nanoparticle.Alternatively, the contrast agent may be indirectly conjugated to thenanoparticle, by attaching to linkers or chelates. The chelate may beadapted to bind a radionuclide. The chelates that can be attached to thepresent nanoparticle may include, but are not limited to,1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA),diethylenetriaminepentaacetic (DTPA), desferrioxamine (DFO) andtriethylenetetramine (TETA).

Suitable means for imaging, detecting, recording or measuring thepresent nanoparticles may also include, for example, a flow cytometer, alaser scanning cytometer, a fluorescence micro-plate reader, afluorescence microscope, a confocal microscope, a bright-fieldmicroscope, a high content scanning system, and like devices. More thanone imaging techniques may be used at the same time or consecutively todetect the present nanoparticles. In one embodiment, optical imaging isused as a sensitive, high-throughput screening tool to acquire multipletime points in the same subject, permitting semi-quantitativeevaluations of tumor marker levels. This offsets the relativelydecreased temporal resolution obtained with PET, although PET is neededto achieve adequate depth penetration for acquiring volumetric data, andto detect, quantitate, and monitor changes in receptor and/or othercellular marker levels as a means of assessing disease progression orimprovement, as well as stratifying patients to suitable treatmentprotocols.

Therapeutic Agent

A therapeutic agent may be attached to the fluorescent nanoparticle, forexample, for targeted treatment of a disease. The therapeutic agent maybe delivered to a diseased site in a highly specific or localized mannerwith release of the therapeutic agent in the disease site.Alternatively, the therapeutic agent may not be released. Thefluorescent nanoparticle conjugated with the ligand can be used fortargeted delivery of a therapeutic agent to a desired location in avariety of systems, such as on, or within, a cell or cell component,within the body of an organism, such as a human, or across theblood-brain barrier.

The therapeutic agent may be attached to the nanoparticle directly orindirectly. The therapeutic agent can be absorbed into the intersticesor pores of the silica shell, or coated onto the silica shell of thefluorescent nanoparticle. In other embodiments where the silica shell isnot covering the entire surface, the therapeutic agent can be associatedwith the fluorescent core, such as by physical absorption or by bondinginteraction. The therapeutic agent may be associated with the ligandthat is attached to the fluorescent nanoparticle. The therapeutic agentmay also be associated with the organic polymer or the contrast agent.For example, the therapeutic agent may be attached to the nanoparticlethrough PEG. The PEGs can have multiple functional groups for attachmentto the nanoparticle and therapeutic agent. The particle can havedifferent types of functionalized PEGs bearing different functionalgroups that can be attached to multiple therapeutic agents. Thetherapeutic agent may be attached to the nanoparticle covalently ornon-covalently.

As used herein, the term “therapeutic agent” refers to a substance thatmay be used in the diagnosis, cure, mitigation, treatment, or preventionof disease in a human or another animal. Such therapeutic agents includesubstances recognized in the official United States Pharmacopeia,official Homeopathic Pharmacopeia of the United States, officialNational Formulary, or any supplement thereof.

Therapeutic agents that can be incorporated with the fluorescentnanoparticles or the ligated-fluorescent nanoparticles of the inventioninclude nucleosides, nucleoside analogs, small interfering RNA (siRNA),microRNA, oligopeptides, polypeptides, antibodies, COX-2 inhibitors,apoptosis promoters, urinary tract agents, vaginal agents, vasodilatorsneurodegenerative agents (e.g., Parkinson's disease), obesity agents,ophthalmic agents, osteoporosis agents, para-sympatholytics,para-sympathometics, antianesthetics, prostaglandins, psychotherapeuticagents, respiratory agents, sedatives, hypnotics, skin and mucousmembrane agents, anti-bacterials, anti-fungals, antineoplastics,cardioprotective agents, cardiovascular agents, anti-thrombotics,central nervous system stimulants, cholinesterase inhibitors,contraceptives, dopamine receptor agonists, erectile dysfunction agents,fertility agents, gastrointestinal agents, gout agents, hormones,immunomodulators, suitably functionalized analgesics or general or localanesthetics, anti-convulsants, anti-diabetic agents, anti-fibroticagents, anti-infectives, motion sickness agents, muscle relaxants,immuno-suppressive agents, migraine agents, non-steroidalanti-inflammatory drugs (NSAIDs), smoking cessation agents, orsympatholytics (see Physicians' Desk Reference, 55th ed., 2001, MedicalEconomics Company, Inc., Montvale, N.J., pages 201-202).

Therapeutic agents that may be attached to the present nanoparticleinclude, but are not limited to, DNA alkylating agents, topoisomeraseinhibitors, endoplasmic reticulum stress inducing agents, a platinumcompound, an antimetabolite, vincalkaloids, taxanes, epothilones, enzymeinhibitors, receptor antagonists, therapeutic antibodies, tyrosinekinase inhibitors, boron radiosensitizers (i.e. velcade), andchemotherapeutic combination therapies.

Non-limiting examples of DNA alkylating agents are nitrogen mustards,such as Mechlorethamine, Cyclophosphamide (Ifosfamide, Trofosfamide),Chlorambucil (Melphalan, Prednimustine), Bendamustine, Uramustine andEstramustine; nitrosoureas, such as Carmustine (BCNU), Lomustine(Semustine), Fotemustine, Nimustine, Ranimustine and Streptozocin; alkylsulfonates, such as Busulfan (Mannosulfan, Treosulfan); Aziridines, suchas Carboquone, ThioTEPA, Triaziquone, Triethylenemelamine; Hydrazines(Procarbazine); Triazenes such as Dacarbazine and Temozolomide;Altretamine and Mitobronitol.

Non-limiting examples of Topoisomerase I inhibitors include Campothecinderivatives including CPT-11 (irinotecan), SN-38, APC, NPC, campothecin,topotecan, exatecan mesylate, 9-nitrocamptothecin, 9-aminocamptothecin,lurtotecan, rubitecan, silatecan, gimatecan, diflomotecan, extatecan,BN-80927, DX-8951f, and MAG-CPT as decribed in Pommier Y. (2006) Nat.Rev. Cancer 6(10):789-802 and U.S. Patent Publication No. 200510250854;Protoberberine alkaloids and derivatives thereof including berberrubineand coralyne as described in Li et al. (2000) Biochemistry39(24):7107-7116 and Gatto et al. (1996) Cancer Res. 15(12):2795-2800;Phenanthroline derivatives including Benzo[i]phenanthridine, Nitidine,and fagaronine as described in Makhey et al. (2003) Bioorg. Med. Chem.11 (8): 1809-1820; Terbenzimidazole and derivatives thereof as describedin Xu (1998) Biochemistry 37(10):3558-3566; and Anthracyclinederivatives including Doxorubicin, Daunorubicin, and Mitoxantrone asdescribed in Foglesong et al. (1992) Cancer Chemother. Pharmacol.30(2):123-]25, Crow et al. (1994) J. Med. Chem. 37(19):31913194, andCrespi et al. (1986) Biochem. Biophys. Res. Commun. 136(2):521-8.Topoisomerase II inhibitors include, but are not limited to Etoposideand Teniposide. Dual topoisomerase I and II inhibitors include, but arenot limited to, Saintopin and other Naphthecenediones, DACA and otherAcridine-4-Carboxamindes, Intoplicine and other Benzopyridoindoles,TAS-I03 and other 7H-indeno[2,1-c]Quinoline-7-ones, Pyrazoloacridine, XR11576 and other Benzophenazines, XR 5944 and other Dimeric compounds,7-oxo-7H-dibenz[f,ij]Isoquinolines and 7-oxo-7H-benzo[e]Perimidines, andAnthracenyl-amino Acid Conjugates as described in Denny and Baguley(2003) Curr. Top. Med. Chem. 3(3):339-353. Some agents inhibitTopoisomerase II and have DNA intercalation activity such as, but notlimited to, Anthracyclines (Aclarubicin, Daunorubicin, Doxorubicin,Epirubicin, Idarubicin, Amrubicin, Pirarubicin, Valrubicin, Zorubicin)and Antracenediones (Mitoxantrone and Pixantrone).

Examples of endoplasmic reticulum stress inducing agents include, butare not limited to, dimethyl-celecoxib (DMC), nelfinavir, celecoxib, andboron radiosensitizers (i.e. velcade (Bortezomib)).

Non-limiting examples of platinum based compound include Carboplatin,Cisplatin, Nedaplatin, Oxaliplatin, Triplatin tetranitrate, Satraplatin,Aroplatin, Lobaplatin, and JM-216. (see McKeage et al. (1997) J. Clin.Oncol. 201:1232-1237 and in general, CHEMOTHERAPY FOR GYNECOLOGICALNEOPLASM, CURRENT THERAPY AND NOVEL APPROACHES, in the Series Basic andClinical Oncology, Angioli et al. Eds., 2004).

Non-limiting examples of antimetabolite agents include Folic acid based,i.e. dihydrofolate reductase inhibitors, such as Aminopterin,Methotrexate and Pemetrexed; thymidylate synthase inhibitors, such asRaltitrexed, Pemetrexed; Purine based, i.e. an adenosine deaminaseinhibitor, such as Pentostatin, a thiopurine, such as Thioguanine andMercaptopurine, a halogenated/ribonucleotide reductase inhibitor, suchas Cladribine, Clofarabine, Fludarabine, or a guanine/guanosine:thiopurine, such as Thioguanine; or Pyrimidine based, i.e.cytosine/cytidine: hypomethylating agent, such as Azacitidine andDecitabine, a DNA polymerase inhibitor, such as Cytarabine, aribonucleotide reductase inhibitor, such as Gemcitabine, or athymine/thymidine: thymidylate synthase inhibitor, such as aFluorouracil (5-FU). Equivalents to 5-FU include prodrugs, analogs andderivative thereof such as 5′-deoxy-5-fluorouridine (doxifluroidine),1-tetrahydrofuranyl-5-fluorouracil (ftorafur), Capecitabine (Xeloda),S-I (MBMS-247616, consisting of tegafur and two modulators, a5-chloro-2,4dihydroxypyridine and potassium oxonate), ralititrexed(tomudex), nolatrexed (Thymitaq, AG337), LY231514 and ZD9331, asdescribed for example in Papamicheal (1999) The Oncologist 4:478-487.

Examples of vincalkaloids, include, but are not limited to Vinblastine,Vincristine, Vinflunine, Vindesine and Vinorelbine.

Examples of taxanes include, but are not limited to docetaxel,Larotaxel, Ortataxel, Paclitaxel and Tesetaxel. An example of anepothilone is iabepilone.

Examples of enzyme inhibitors include, but are not limited tofarnesyltransferase inhibitors (Tipifamib); CDK inhibitor (Alvocidib,Seliciclib); proteasome inhibitor (Bortezomib); phosphodiesteraseinhibitor (Anagrelide; rolipram); IMP dehydrogenase inhibitor(Tiazofurine); and lipoxygenase inhibitor (Masoprocol). Examples ofreceptor antagonists include, but are not limited to ERA (Atrasentan);retinoid X receptor (Bexarotene); and a sex steroid (Testolactone).

Examples of therapeutic antibodies include, but are not limited toanti-HER1/EGFR (Cetuximab, Panitumumab); Anti-HER2/neu (erbB2) receptor(Trastuzumab); Anti-EpCAM (Catumaxomab, Edrecolomab) Anti-VEGF-A(Bevacizumab); Anti-CD20 (Rituximab, Tositumomab, Ibritumomab);Anti-CD52 (Alemtuzumab); and Anti-CD33 (Gemtuzumab). U.S. Pat. Nos.5,776,427 and 7,601,355.

Examples of tyrosine kinase inhibitors include, but are not limited toinhibitors to ErbB: HER1/EGFR (Erlotinib, Gefitinib, Lapatinib,Vandetanib, Sunitinib, Neratinib); HER2/neu (Lapatinib, Neratinib); RTKclass III: C-kit (Axitinib, Sunitinib, Sorafenib), FLT3 (Lestaurtinib),PDGFR (Axitinib, Sunitinib, Sorafenib); and VEGFR (Vandetanib,Semaxanib, Cediranib, Axitinib, Sorafenib); bcr-abl (Imatinib,Nilotinib, Dasatinib); Src (Bosutinib) and Janus kinase 2(Lestaurtinib).

Chemotherapeutic agents that can be attached to the present nanoparticlemay also include amsacrine, Trabectedin, retinoids (Alitretinoin,Tretinoin), Arsenic trioxide, asparagine depleterAsparaginase/Pegaspargase), Celecoxib, Demecolcine, Elesclomol,Elsamitrucin, Etoglucid, Lonidamine, Lucanthone, Mitoguazone, Mitotane,Oblimersen, Temsirolimus, and Vorinostat.

Examples of specific therapeutic agents that can be linked, ligated, orassociated with the fluorescent nanoparticles of the invention areflomoxef; fortimicin(s); gentamicin(s); glucosulfone solasulfone;gramicidin S; gramicidin(s); grepafloxacin; guamecycline; hetacillin;isepamicin; josamycin; kanamycin(s); flomoxef; fortimicin(s);gentamicin(s); glucosulfone solasulfone; gramicidin S; gramicidin(s);grepafloxacin; guamecycline; hetacillin; isepamicin; josamycin;kanamycin(s); bacitracin; bambermycin(s); biapenem; brodimoprim;butirosin; capreomycin; carbenicillin; carbomycin; carumonam;cefadroxil; cefamandole; cefatrizine; cefbuperazone; cefclidin;cefdinir; cefditoren; cefepime; cefetamet; cefixime; cefinenoxime;cefininox; cladribine; apalcillin; apicycline; apramycin; arbekacin;aspoxicillin; azidamfenicol; aztreonam; cefodizime; cefonicid;cefoperazone; ceforamide; cefotaxime; cefotetan; cefotiam; cefozopran;cefpimizole; cefpiramide; cefpirome; cefprozil; cefroxadine; cefteram;ceftibuten; cefuzonam; cephalexin; cephaloglycin; cephalosporin C;cephradine; chloramphenicol; chlortetracycline; clinafloxacin;clindamycin; clomocycline; colistin; cyclacillin; dapsone;demeclocycline; diathymosulfone; dibekacin; dihydrostreptomycin;6-mercaptopurine; thioguanine; capecitabine; docetaxel; etoposide;gemcitabine; topotecan; vinorelbine; vincristine; vinblastine;teniposide; melphalan; methotrexate; 2-p-sulfanilyanilinoethanol;4,4′-sulfinyldianiline; 4-sulfanilamidosalicylic acid; butorphanol;nalbuphine. streptozocin; doxorubicin; daunorubicin; plicamycin;idarubicin; mitomycin C; pentostatin; mitoxantrone; cytarabine;fludarabine phosphate; butorphanol; nalbuphine. streptozocin;doxorubicin; daunorubicin; plicamycin; idarubicin; mitomycin C;pentostatin; mitoxantrone; cytarabine; fludarabine phosphate;acediasulfone; acetosulfone; amikacin; amphotericin B; ampicillin;atorvastatin; enalapril; ranitidine; ciprofloxacin; pravastatin;clarithromycin; cyclosporin; famotidine; leuprolide; acyclovir;paclitaxel; azithromycin; lamivudine; budesonide; albuterol; indinavir;metformin; alendronate; nizatidine; zidovudine; carboplatin; metoprolol;amoxicillin; diclofenac; lisinopril; ceftriaxone; captopril; salmeterol;xinafoate; imipenem; cilastatin; benazepril; cefaclor; ceftazidime;morphine; dopamine; bialamicol; fluvastatin; phenamidine; podophyllinicacid 2-ethylhydrazine; acriflavine; chloroazodin; arsphenamine;amicarbilide; aminoquinuride; quinapril; oxymorphone; buprenorphine;floxuridine; dirithromycin; doxycycline; enoxacin; enviomycin;epicillin; erythromycin; leucomycin(s); lincomycin; lomefloxacin;lucensomycin; lymecycline; meclocycline; meropenem; methacycline;micronomicin; midecamycin(s); minocycline; moxalactam; mupirocin;nadifloxacin; natamycin; neomycin; netilmicin; norfloxacin;oleandomycin; oxytetracycline; p-sulfanilylbenzylamine; panipenem;paromomycin; pazufloxacin; penicillin N; pipacycline; pipemidic acid;polymyxin; primycin; quinacillin; ribostamycin; rifamide; rifampin;rifamycin SV; rifapentine; rifaximin; ristocetin; ritipenem;rokitamycin; rolitetracycline; rosaramycin; roxithromycin;salazosulfadimidine; sancycline; sisomicin; sparfloxacin; spectinomycin;spiramycin; streptomycin; succisulfone; sulfachrysoidine; sulfaloxicacid; sulfamidochrysoidine; sulfanilic acid; sulfoxone; teicoplanin;temafloxacin; temocillin; tetroxoprim; thiamphenicol; thiazolsulfone;thiostrepton; ticarcillin; tigemonam; tobramycin; tosufloxacin;trimethoprim; trospectomycin; trovafloxacin; tuberactinomycin;vancomycin; azaserine; candicidin(s); chlorphenesin; dermostatin(s);filipin; fungichromin; mepartricin; nystatin; oligomycin(s); perimycinA; tubercidin; 6-azauridine; 6-diazo-5-oxo-L-norleucine;aclacinomycin(s); ancitabine; anthramycin; azacitadine; azaserine;bleomycin(s); ethyl biscoumacetate; ethylidene dicoumarol; iloprost;lamifiban; taprostene; tioclomarol; tirofiban; amiprilose; bucillamine;gusperimus; gentisic acid; glucamethacin; glycol salicylate;meclofenamic acid; mefenamic acid; mesalamine; niflumic acid;olsalazine; oxaceprol; S-enosylmethionine; salicylic acid; salsalate;sulfasalazine; tolfenamic acid; carubicin; carzinophillin A;chlorozotocin; chromomycin(s); denopterin; doxifluridine; edatrexate;eflornithine; elliptinium; enocitabine; epirubicin; mannomustine;menogaril; mitobronitol; mitolactol; mopidamol; mycophenolic acid;nogalamycin; olivomycin(s); peplomycin; pirarubicin; piritrexim;prednimustine; procarbazine; pteropterin; puromycin; ranimustine;streptonigrin; thiamiprine; mycophenolic acid; procodazole; romurtide;sirolimus (rapamycin); tacrolimus; butethamine; fenalcomine;hydroxytetracaine; naepaine; orthocaine; piridocaine; salicyl alcohol;3-amino-4-hydroxybutyric acid; aceclofenac; alminoprofen; amfenac;bromfenac; bromosaligenin; bumadizon; carprofen; diclofenac; diflunisal;ditazol; enfenamic acid; etodolac; etofenamate; fendosal; fepradinol;flufenamic acid; Tomudex®(N-[[5-[[(1,4-Dihydro-2-methyl-4-oxo-6-quin-azolinyl)methyl]methylamino]-2-thienyl]carbonyl]-glutamicacid), trimetrexate, tubercidin, ubenimex, vindesine, zorubicin;argatroban; coumetarol or dicoumarol.

Lists of additional therapeutic agents can be found, for example, in:Physicians' Desk Reference, 55th ed., 2001, Medical Economics Company,Inc., Montvale, N.J.; USPN Dictionary of USAN and International DrugNames, 2000, The United States Pharmacopeial Convention, Inc.,Rockville, Md.; and The Merck Index, 12th ed., 1996, Merck & Co., Inc.,Whitehouse Station, N.J.

The therapeutic agent may also include radionuclides when the presentnanoparticle is used in targeted radiotherapy. In one embodiment, lowenergy beta-emitting radionuclides, such as ¹⁷⁷Lu-chelated constructs,is associated with the nanoparticle and used to treat relatively smalltumor burdens or micrometastatic disease. In another embodiment, higherenergy beta emitters, such as yttrium-90 (⁹⁰Y), may be used to treatlarger tumor burdens. Iodine-131 (¹³¹I) may also be used forradiotherapy.

The surface of the nanoparticle may be modified to incorporate at leastone functional group. The organic polymer (e.g., PEG) attached to thenanoparticle may be modified to incorporate at least one functionalgroup. For example, the functional group can be a maleimide orN-Hydroxysuccinimide (NETS) ester. The incorporation of the functionalgroup makes it possible to attach various ligands, contrast agentsand/or therapeutic agents to the nanoparticle.

In one embodiment, a therapeutic agent is attached to the nanoparticle(surface or the organic polymer coating) via an NHS ester functionalgroup. For example, tyrosine kinase inhibitor such as dasatinib (BMS) orchemotherapeutic agent (e.g., taxol), can be coupled via an ester bondto the nanoparticle. This ester bond can then be cleaved in an acidicenvironment or enzymatically in vivo. This approach may be used todeliver a prodrug to a subject where the drug is released from theparticle in vivo.

We have tested the prodrug approach by coupling small molecule inhibitordasatinib with the PEG molecules of the nanoparticle. Based onbiodistribution results and the human drug dosing calculations, thenanoparticle has been found to have unique biological properties,including relatively rapid clearance from the blood compared to tumorsand subsequent tumor tissue accumulation of the therapeutic agent, whichsuggests that a prodrug approach is feasible. The functionalizednanoparticle permits drugs to be dosed multiple times, ensuring that thedrug concentration in the tumor is greater than that specified by theIC-50 in tumor tissue, yet will not be dose-limiting to other organtissues, such as the heart, liver or kidney. The therapeutic agent andnanoparticle can be radiolabeled or optically labelled separately,allowing independent monitoring of the therapeutic agent and thenanoparticle. In one embodiment, radiofluorinated (i.e., ¹⁸F) dasatinibis coupled with PEG-3400 moieties attached to the nanoparticle via NHSester linkages. Radiofluorine is crucial for being able to independentlymonitor time-dependent changes in the distribution and release of thedrug from the radioiodinated (¹²⁴I) fluorescent (Cy5) nanoparticle. Inthis way, we can separately monitor the prodrug (dasatinib) andnanoparticle. This permits optimization of the prodrug design comparedwith methods in the prior art where no dual-labeling approach is used.In another embodiment, radiotherapeutic iodine molecules (i.e., I-131),or other therapeutic gamma or alpha emitters, are conjugated with PEGvia a maleimide functional group, where the therapeutic agent may notdissociate from the PEG in vivo.

A generalizable approach referred herein as “click chemistry” isdescribed below. In order for the present nanoparticle to readilyaccommodate large ranges of ligands, contrast agents or chelates, thesurface of the nanoparticle may be modified to incorporate a functionalgroup. The nanoparticle may also be modified with organic polymers(e.g., PEGs) or chelates that can incorporate a functional group. In themeantime, the ligand, contrast agent, or therapeutic agent is modifiedto incorporate a functional group that is able to react with thefunctional group on the nanoparticle, or on the PEGs or chelating agentsattached to the nanoparticle under suitable conditions. Accordingly, anyligand, contrast agent or therapeutic agent that has the reactivefunctional group is able to be readily conjugated to the nanoparticle.This generalizable approach is referred herein as “click chemistry”,which would allow for a great deal of versatility to exploremultimodality applications. In the chemical reactions of “clickchemistry”, two molecular components may be joined via a selective,rapid, clean, bioorthogonal, and/or biocompatible reaction. Kolb et al.,(2001) Click Chemistry: Diverse Chemical Function from a Few GoodReactions. Angewandte Chemie International Edition 40, 2004-2021. Lim etal., (2010) Bioorthogonal chemistry: recent progress and futuredirections. Chemical Communications 46, 1589-1600. Sletten et al.,(2009) Bioorthogonal chemistry: fishing for selectivity in a sea offunctionality. Angewandte Chemie International Edition 48, 6973-6998.

Any suitable reaction mechanism may be adapted in the present inventionfor “click chemistry”, so long as facile and controlled attachment ofthe ligand, contrast agent or chelate to the nanoparticle can beachieved. In one embodiment, a free triple bond is introduced onto PEG,which is already covalently conjugated with the shell of thenanoparticle. In the meantime, an azide bond is introduced onto thedesired ligand (or contrast agent, chelate). When the PEGylatednanoparticle and the ligand (or contrast agent, chelate) are mixed inthe presence of a copper catalyst, cycloaddition of azide to the triplebond will occur, resulting in the conjugation of the ligand with thenanoparticle. One example of click chemistry is the Cu(I)-catalyzed[3+2] Huisgen cycloaddition between an azide and alkyne. Moses et al.,(2007) The growing applications of click chemistry. Chemical SocietyReviews 36, 1249-1262. Glaser et al., (2009) ‘Click labelling’ in PETradiochemistry. Journal of Labelled Compounds & Radiopharmaceuticals 52,407-414. Mindt et al., (2009) A “Click Chemistry” Approach to theEfficient Synthesis of Multiple Imaging Probes Derived from a SinglePrecursor. Bioconjugate Chemistry 20, 1940-1949. New et al., (2009)Growing applications of “click chemistry” for bioconjugation incontemporary biomedical research. Cancer Biotherapy andRadiopharmaceuticals 24, 289-301. Wang et al., (2010) Application of“Click Chemistry” in Synthesis of Radiopharmaceuticals. Progress inChemistry 22, 1591-1602. Schultz et al., (2010) Synthesis of aDOTA-Biotin Conjugate for Radionuclide Chelation via Cu-Free ClickChemistry. Organic Letters 12, 2398-2401. Martin et al., (2010) ADOTA-peptide conjugate by copper-free click chemistry. Bioorganic &Medicinal Chemistry Letters 20, 4805-4807. Lebedev et al., (2009)Clickable bifunctional radiometal chelates for peptide labeling.Chemical Communications 46, 1706-1708. Knor et al., (2007) Synthesis ofnovel 1,4,7,10-tetraazacyclodecane-1,4,7,10-tetraacetic acid (DOTA)derivatives for chemoselective attachment to unprotectedpolyfunctionalized compounds. Chemistry—a European Journal 13,6082-6090. In a second embodiment, a maleimide functional group and athiol group may be introduced onto the nanoparticle and the desiredligand (or contrast agent, chelate), with the nanoparticle having themaleimide functional group, the ligand (or contrast agent, chelate)having the thiol group, or vice versa. The double bond of maleimidereadily reacts with the thiol group to form a stable carbon-sulfur bond.In a third embodiment, an activated ester functional group, e.g., asuccinimidyl ester group, and an amine group may be introduced onto thenanoparticle and the desired ligand, contrast agent or chelate. Theactivated ester group readily reacts with the amine group to form astable carbon-nitrogen amide bond. In a fourth embodiment, the clickchemistry involves the inverse electron demand Diels-Alder reactionbetween a tetrazine moiety and a strained alkene (FIG. 21C). Devaraj etal., (2009) Fast and Sensitive Pretargeted Labeling of Cancer Cellsthrough a Tetrazine/trans-Cyclooctene Cycloaddition. AngewandteChemie-International Edition 48, 7013-7016. Devaraj et al., (2008)Tetrazine-Based Cycloadditions: Application to Pretargeted Live CellImaging. Bioconjugate Chemistry 19, 2297-2299. Blackman et al., (2008)Tetrazine ligation: fast bioconjugation based on inverse electron demandDiels-Alder reactivity. Journal of the American Chemical Society 130,13518-13519. The ligation can be selective, fast, biocompatible, and/orbioorthogonal. Unlike many Diels-Alder reactions, the coupling isirreversible, forming a stable pyridazine products after theretro-Diels-Alder release of dinitrogen from the reaction intermediate.A tetrazine moiety and a strained alkene may be introduced onto thenanoparticle and the desired ligand (or contrast agent, chelate), withthe nanoparticle having the tetrazine moiety, the ligand (or contrastagent, chelate) having the strained alkene, or vice versa. A number ofdifferent tetrazine-strained alkene pairs can be used for the reaction,including, e.g., the combination of 3-(4-benzylamino)-1,2,4,5-tetrazine(Tz) and either norbornene- or trans-cyclooctene-dervatives. Schoch etal., (2010) Post-Synthetic Modification of DNA byInverse-Electron-Demand Diels-Alder Reaction. Journal of the AmericanChemical Society 132, 8846-8847. Devaraj et al., (2010) BioorthogonalTurn-On Probes for Imaging Small Molecules Inside Living Cells.Angewandte Chemie International Edition 49. Haun et al., (2010)Bioorthogonal chemistry amplifies nanoparticle binding and enhances thesensitivity of cell detection. Nature Nanotechnology 5, 660-665. Rossinet al., (2010) In vivo chemisry for pretargeted tumor imaging in livemice. Angewandte Chemie International Edition 49, 3375-3378. Li et al.,(2010) Tetrazine-trans-cyclooctene ligation for the rapid constructionof 18-F labeled probes. Chemical Communications 46, 8043-8045. Reiner etal., (2011) Synthesis and in vivo imaging of a 18F-labeled PARP1inhibitor using a chemically orthogonal scavenger-assistedhigh-performance method. Angewandte Chemie International Edition 50,1922-1925. For example, the surface of the nanoparticles may bedecorated with tetrazine moieties, which can subsequently be conjugatedto norbornene-modified ligand (or contrast agent, or chelate) (FIGS. 21Dand 21E). In one aspect, the nanoparticles are coated with tetrazine,which can then be modified with the DOTA chelates using thetetrazine-norbornene ligation, and radiolabeled with ⁶⁴Cu.

Residence/Clearance Time In Vivo

After administration of the present nanoparticle to a subject, the bloodresidence half-time of the nanoparticles may range from about 2 hours toabout 25 hours, from about 3 hours to about 20 hours, from about 3 hoursto about 15 hours, from about 4 hours to about 10 hours, or from about 5hours to about 6 hours. Longer blood residence half-time means longercirculation, which allows more nanoparticles to accumulate at the targetsite in vivo. Blood residence half-time may be evaluated as follows. Thenanoparticles are first administered to a subject (e.g., a mouse, aminiswine or a human). At various time points post administration, bloodsamples are taken to measure nanoparticle concentrations throughsuitable methods.

In one embodiment, after administration of the PEGylated (or control)nanoparticle to a subject, blood residence half-time of the nanoparticlemay range from about 2 hours to about 15 hours, or from about 4 hours toabout 10 hours. Tumor residence half-time of the nanoparticle afteradministration of the nanoparticle to a subject may range from about 5hours to about 2 days, from about 10 hours to about 4 days, or fromabout 15 hours to about 3.5 days. The ratio of tumor residence half-timeto blood residence halftime of the nanoparticle after administration ofthe nanoparticle to a subject may range from about 2 to about 30, fromabout 3 to about 20, or from about 4 to about 15. Renal clearance of thenanoparticle after administration of the nanoparticle to a subject mayrange from about 10% ID (initial dose) to about 100% ID in about 24hours, from about 30% ID to about 80% ID in about 24 hours, or fromabout 40% ID to about 70% ID in about 24 hours. In one embodiment, afterthe nanoparticle is administered to a subject, blood residence half-timeof the nanoparticle ranges from about 2 hours to about 25 hours, tumorresidence half-time of the nanoparticle ranges from about 5 hours toabout 5 days, and renal clearance of the nanoparticle ranges from about30% ID to about 80% ID in about 24 hours.

After administration of the present nanoparticle to a subject, the tumorresidence half-time of the present nanoparticles may range from about 5hours to about 5 days, from about 10 hours to about 4 days, from about15 hours to about 3.5 days, from about 20 hours to about 3 days, fromabout 2.5 days to about 3.1 days, from about 1 day to 3 days, or about73.5 hours.

The ratio of the tumor residence half-time to the blood residencehalf-time of the nanoparticle may range from about 2 to about 30, fromabout 3 to about 20, from about 4 to about 15, from about 4 to about 10,from about 10 to about 15, or about 13.

In one embodiment, the present invention provides a fluorescentsilica-based nanoparticle comprising a silica-based core comprising afluorescent compound positioned within the silica-based core; a silicashell surrounding at least a portion of the core; an organic polymerattached to the nanoparticle; and a ligand attached to the nanoparticle,wherein the nanoparticle has a diameter between about 1 nm and about 15nm. After administration of the PEGylated (control) nanoparticle to asubject, blood residence half-time of the nanoparticle may range fromabout 2 hours to about 25 hours, or from about 2 hours to about 15hours; tumor residence half-time of the nanoparticle may range fromabout 5 hours to about 2 days; and renal clearance of the nanoparticlemay range from about 30% ID to about 80% ID in about 24 hours. Thenumber of ligands attached to the nanoparticle may range from about 1 toabout 30, or from about 1 to about 10. The diameter of the nanoparticlemay be between about 1 nm and about 8 nm. A contrast agent, such as aradionuclide, may be attached to the nanoparticle. Alternatively, achelate may be attached to the nanoparticle. The nanoparticle may bedetected by PET, SPECT, CT, MM, optical imaging, bioluminescenceimaging, or combinations thereof. A therapeutic agent may be attached tothe nanoparticle. After administration of the radiolabeled targetednanoparticle to a subject, blood residence half-time of the nanoparticlemay also range from about 3 hours to about 15 hours, or from about 4hours to about 10 hours. Tumor residence half-time of the nanoparticleafter administration of the nanoparticle to a subject may also rangefrom about 10 hours to about 4 days, or from about 15 hours to about 3.5days. The ratio of tumor residence half-time to blood residencehalf-time of the targeted nanoparticle after administration of thenanoparticle to a subject may range from about 2 to about 30, from about3 to about 20, or from about 4 to about 15. Renal clearance of thenanoparticle may also range from about 45% ID to about 90% ID in about24 hours after administration of the nanoparticle to a subject.

In one embodiment, to estimate residence (or clearance) half-time valuesof the radiolabeled nanoparticles (T_(1/2)) in blood, tumor, and othermajor organs/tissues, the percentage of the injected dose per gram (%ID/g) values are measured by sacrificing groups of mice at specifiedtimes following administration of the nanoparticles. Blood, tumor, andorgans are harvested, weighed, and counted in a scintillation γ-counter.The % ID/g values are corrected for radioactive decay to the time ofinjection. The resulting time-activity concentration data for eachtissue are fit to a decreasing monoexponential function to estimatetissue/organ T_(1/2) values.

After administration of the present nanoparticle to a subject, the renalclearance of the present nanoparticles may range from about 45% ID(initial dose) to greater than 90% ID in about 24 hours, from about 20%ID to about 90% ID in about 24 hours, from about 30% ID to about 80% IDin about 24 hours, from about 40% ID to about 70% ID in about 24 hours,from about 40% ID to about 60% ID in about 24 hours, from about 40% IDto about 50% ID in about 24 hours, about 43% ID in about 24 hours, fromabout 10% ID to about 100% ID in about 24 hours, from about 40% ID toabout 100% ID in about 24 hours, from about 80% ID to about 100% ID inabout 24 hours, from about 90% ID to about 95% ID in about 24 hours,from about 90% ID to about 100% ID in about 24 hours, or from about 80%ID to about 95% ID in about 24 hours. Renal clearance may be evaluatedas follows. The nanoparticles are first administered to a subject (e.g.,a mouse, a miniswine or a human). At various time points postadministration, urine samples are taken to measure nanoparticleconcentrations through suitable methods.

In one embodiment, renal clearance (e.g., the fraction of nanoparticlesexcreted in the urine over time) is assayed as follows. A subject isadministered with the present nanoparticles, and urine samples collectedover a certain time period (e.g., 168 hours). Particle concentrations ateach time point are determined using fluorometric analyses and a serialdilution calibration curve generated from background-correctedfluorescence signal measurements of urine samples mixed with knownparticle concentrations (% ID). Concentration values, along withestimates of average daily mouse urine volumes, are used to computecumulative % ID/g urine excreted. In another embodiment, renal clearanceof radiolabeled nanoparticles is assayed by measuring urine specimenactivities (counts per minute) over similar time intervals using, forexample, γ-counting, and after nanoparticle administration to computecumulative urine excretion.

In a third embodiment, to assess cumulative fecal excretion, feces arecollected in metabolic cages over similar time intervals afteradministration of the nanoparticles and specimen activities determinedusing a γ-counter.

When the nanoparticles in the amount of about 100 times of the humandose equivalent are administered to a subject, substantially no anemia,weight loss, agitation, increased respiration, GI disturbance, abnormalbehavior, neurological dysfunction, abnormalities in hematology,abnormalities in clinical chemistries, drug-related lesions in organpathology, mortality, or combination thereof are observed in about 10 toabout 14 days.

When the present nanoparticle contains at least one attached ligand, themultivalency enhancement of the nanoparticle (e.g., compared to theligand alone) may range from about 1.5 fold to about 10 fold, from about2 fold to about 8 fold, from about 2 fold to about 6 fold, from about 2fold to about 4 fold, or about 2 fold.

The nanoparticles of the present invention show unexpected in vitro andin vivo physicochemical and biological parameters in view of the priorart. For example, the blood residence half-time estimated for theligand-attached nanoparticles (e.g., about 5.5 hrs for cRGD-attachednanoparticles) is substantially longer than that of the correspondingligand (e.g., about 13 minutes for cRGD). Montet et al. Multivalenteffects of RGD peptides obtained by nanoparticle display. J Med Chem.49, 6087-6093 (2006). Extended blood residence half-times may enhanceprobe bioavailability, facilitate tumor targeting, and yield highertumor uptake over longer time periods. In one embodiment, the tumorresidence half-time for the targeted nanoparticles (i.e.,ligand-attached nanoparticles) is about 13 times greater than bloodresidence half-time, whereas the tumor residence half-time for thenon-targeted nanoparticles (i.e., corresponding nanoparticles notattached with ligands) is only about 5 times greater than bloodresidence half-time. This difference suggests substantially greatertumor tissue accumulation of the targeted nanoparticles compared withthe non-targeted nanoparticles. In certain embodiments, given the numberof ligands attached to the nanoparticle, the present nanoparticles showunexpected high-affinity binding (e.g., K_(d) 0.51 nM and IC₅₀ 1.2 nMfor cRGD-attached nanoparticle), multivalency enhancement (e.g., morethan 2 fold enhancement for cRGD-attached nanoparticles compared to cRGDpeptide alone), significant differential tumor uptake (e.g.,cRGD-attached PEG-nanoparticles show about 3 to 4 fold increase indifferential tumor uptake relative to the PEG-coated nanoparticles over72 hrs post-administration), and significant tumor contrast relative tonormal muscle (e.g., about 3 to 5 fold over 72 hrs post-administration)based on tumor-to-muscle uptake ratios.

In one embodiment, three-fold activity-concentration increases werefound for ligand-attached nanoparticles in integrin-expressing tumorsover controls (e.g., ligand-attached nanoparticles in non-integrinexpressing tumors, or corresponding nanoparticles not attached withligands in integrin-expressing tumors) at the time of maximum tumoruptake (about 4 hrs post-injection of the nanoparticles). In addition,tumor-to-muscle uptake ratios for targeted nanoparticles (i.e.,ligand-attached nanoparticles) reveal enhanced tumor tissue contrastrelative to normal muscle, compared with decreased tumor tissue contrastrelative to normal muscle for non-targeted nanoparticles (i.e.,corresponding nanoparticles not attached with ligands), suggesting thatthe targeted nanoparticles are tumor-selective.

In another embodiment, the targeted and non-targeted nanoparticles bothshow efficient renal excretion over the same time period. Nearly half ofthe injected dose is excreted over the first 24 hrs post-injection andabout 72% by 96 hrs, suggesting that the bulk of excretion occurred inthe first day post-injection. By contrast, fecal excretion profiles ofthe targeted nanoparticles indicate that, on average, 7% and 15% of theinjected dose is eliminated over 24 and 96 hrs, respectively.

The physicochemical and biological parameters of the non-toxicnanoparticles, along with its multimodal imaging capabilities (e.g., PETand optical imaging), expand the range of their potential biomedicalapplications. The applications include (a) long-term monitoring: theextended blood circulation time and corresponding bioavailability of thenanoparticles highlight their versatility for both early and long-termmonitoring of various stages of disease management (such as diagnosticscreening, pre-treatment evaluation, therapeutic intervention, andpost-treatment monitoring) without restrictions imposed by toxicityconsiderations; (b) improved tumor penetration: the clearance propertiesof the targeted nanoparticles (e.g., their renal clearance is slowerthat of the molecular probes in the prior art) will be useful forvarious types of biological applications. For example, the nanoparticleswould be particularly useful in cases of poorly vascularized andrelatively inaccessible solid tumors in which localization of agents istypically slow after systemic administration; (c) multimodal imagingcapabilities: these modalities can be combined at multiple scales (i.e.,whole body to cellular levels) for acquiring complementary,depth-sensitive biological information. For example, in SLN mapping,deep nodes can be mapped by PET in terms of their distribution andnumber, while more precise and detailed localization of superficialnodes can be obtained by fluorescence imaging; and (d) targetedtherapeutics: longer clearance of the targeted nanoparticles from tumorcompared to that from blood may be exploited for combineddiagnostic/therapeutic applications, in which the nanoparticles canserve as a radiotherapeutic or drug delivery vehicle.

Pharmaceutical Compositions

The present invention further provides a pharmaceutical compositioncomprising the present nanoparticle. The pharmaceutical compositions ofthe invention may be administered orally in the form of a suitablepharmaceutical unit dosage form. The pharmaceutical compositions of theinvention may be prepared in many forms that include tablets, hard orsoft gelatin capsules, aqueous solutions, suspensions, and liposomes andother slow-release formulations, such as shaped polymeric gels.

Suitable modes of administration for the present nanoparticle orcomposition include, but are not limited to, oral, intravenous, rectal,sublingual, mucosal, nasal, ophthalmic, subcutaneous, intramuscular,transdermal, intradermal, subdermal, peritumoral, spinal, intrathecal,intra-articular, intra-arterial, sub-arachnoid, bronchial, and lymphaticadministration, and other dosage forms for systemic delivery of activeingredients. The present pharmaceutical composition may be administeredby any method known in the art, including, without limitation,transdermal (passive via patch, gel, cream, ointment or iontophoretic);intravenous (bolus, infusion); subcutaneous (infusion, depot);transmucosal (buccal and sublingual, e.g., orodispersible tablets,wafers, film, and effervescent formulations; conjunctival (eyedrops);rectal (suppository, enema)); or intradermal (bolus, infusion, depot).The composition may be delivered topically.

Oral liquid pharmaceutical compositions may be in the form of, forexample, aqueous or oily suspensions, solutions, emulsions, syrups orelixirs, or may be presented as a dry product for constitution withwater or other suitable vehicle before use. Such liquid pharmaceuticalcompositions may contain conventional additives such as suspendingagents, emulsifying agents, non-aqueous vehicles (which may includeedible oils), or preservatives.

The nanoparticle pharmaceutical compositions of the invention may alsobe formulated for parenteral administration (e.g., by injection, forexample, bolus injection or continuous infusion) and may be presented inunit dosage form in ampules, pre-filled syringes, small volume infusioncontainers or multi-dose containers with an added preservative. Thepharmaceutical compositions may take such forms as suspensions,solutions, or emulsions in oily or aqueous vehicles, and may containformulating agents such as suspending, stabilizing and/or dispersingagents. Alternatively, the pharmaceutical compositions of the inventionmay be in powder form, obtained by aseptic isolation of sterile solid orby lyophilization from solution, for constitution with a suitablevehicle, e.g., sterile, pyrogen-free water, before use.

For topical administration to the epidermis, the pharmaceuticalcompositions may be formulated as ointments, creams or lotions, or asthe active ingredient of a transdermal patch. Suitable transdermaldelivery systems are disclosed, for example, in A. Fisher et al. (U.S.Pat. No. 4,788,603), or R. Bawa et al. (U.S. Pat. Nos. 4,931,279;4,668,506; and 4,713,224). Ointments and creams may, for example, beformulated with an aqueous or oily base with the addition of suitablethickening and/or gelling agents. Lotions may be formulated with anaqueous or oily base and will in general also contain one or moreemulsifying agents, stabilizing agents, dispersing agents, suspendingagents, thickening agents, or coloring agents. The pharmaceuticalcompositions can also be delivered via ionophoresis, e.g., as disclosedin U.S. Pat. Nos. 4,140,122; 4,383,529; or 4,051,842.

Pharmaceutical compositions suitable for topical administration in themouth include unit dosage forms such as lozenges comprising apharmaceutical composition of the invention in a flavored base, usuallysucrose and acadia or tragacanth; pastilles comprising thepharmaceutical composition in an inert base such as gelatin and glycerinor sucrose and acacia; mucoadherent gels, and mouthwashes comprising thepharmaceutical composition in a suitable liquid carrier.

For topical administration to the eye, the pharmaceutical compositionscan be administered as drops, gels (S. Chrai et al., U.S. Pat. No.4,255,415), gums (S. L. Lin et al., U.S. Pat. No. 4,136,177) or via aprolonged-release ocular insert (A. S. Michaels, U.S. Pat. No. 3,867,519and H. M. Haddad et al., U.S. Pat. No. 3,870,791).

When desired, the above-described pharmaceutical compositions can beadapted to give sustained release of a therapeutic compound employed,e.g., by combination with certain hydrophilic polymer matrices, e.g.,comprising natural gels, synthetic polymer gels or mixtures thereof.

Pharmaceutical compositions suitable for rectal administration whereinthe carrier is a solid are most preferably presented as unit dosesuppositories. Suitable carriers include cocoa butter and othermaterials commonly used in the art, and the suppositories may beconveniently formed by admixture of the pharmaceutical composition withthe softened or melted carrier(s) followed by chilling and shaping inmolds.

Pharmaceutical compositions suitable for vaginal administration may bepresented as pessaries, tampons, creams, gels, pastes, foams or sprayscontaining, in addition to the nanoparticles and the therapeutic agent,such carriers are well known in the art.

For administration by inhalation, the pharmaceutical compositionsaccording to the invention are conveniently delivered from aninsufflator, nebulizer or a pressurized pack or other convenient meansof delivering an aerosol spray. Pressurized packs may comprise asuitable propellant such as dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol, the dosageunit may be determined by providing a valve to deliver a metered amount.

Alternatively, for administration by inhalation or insufflation, thepharmaceutical compositions of the invention may take the form of a drypowder composition, for example, a powder mix of the pharmaceuticalcomposition and a suitable powder base such as lactose or starch. Thepowder composition may be presented in unit dosage form in, for example,capsules or cartridges or, e.g., gelatin or blister packs from which thepowder may be administered with the aid of an inhalator or insufflator.

For intra-nasal administration, the pharmaceutical compositions of theinvention may be administered via a liquid spray, such as via a plasticbottle atomizer. Typical of these are the Mistometer® (isoproterenolinhaler-Wintrop) and the Medihaler® (isoproterenol inhaler—Riker).

Pharmaceutical compositions of the invention may also contain otheradjuvants such as flavorings, colorings, anti-microbial agents, orpreservatives.

It will be further appreciated that the amount of the pharmaceuticalcompositions required for use in treatment will vary not only with thetherapeutic agent selected but also with the route of administration,the nature of the condition being treated and the age and condition ofthe patient and will be ultimately at the discretion of the attendantphysician or clinician. For evaluations of these factors, see J. F.Brien et al., Europ. J. Clin. Pharmacol., 14, 133 (1978); andPhysicians' Desk Reference, Charles E. Baker, Jr., Pub., MedicalEconomics Co., Oradell, N.J. (41st ed., 1987). Generally, the dosages ofthe therapeutic agent when used in combination with the fluorescentnanoparticles of the present invention can be lower than when thetherapeutic agent is administered alone or in conventionalpharmaceutical dosage forms. The high specificity of the fluorescentnanoparticle for a target site, such as a receptor situated on a cell'ssurface, can provide a relatively highly localized concentration of atherapeutic agent, or alternatively, a sustained release of atherapeutic agent over an extended time period.

The present nanoparticles or compositions can be administered to asubject. The subject can be a mammal, preferably a human. Mammalsinclude, but are not limited to, murines, rats, rabbits, simians,bovines, ovine, swine, canines, feline, farm animals, sport animals,pets, equine, and primates.

Uses of Nanoparticles

The present invention further provides a method for detecting acomponent of a cell comprising the steps of: (a) contacting the cellwith a fluorescent silica-based nanoparticle comprising a silica-basedcore comprising a fluorescent compound positioned within thesilica-based core; a silica shell surrounding at least a portion of thecore; an organic polymer attached to the nanoparticle; from about 1 toabout 25 ligands (from about 1 to about 20 ligands, or from about 1 toabout 10 ligands, or other ranges; see discussions herewithin) attachedto the nanoparticle; and a contrast agent or a chelate attached to thenanoparticle; and (b) monitoring the binding of the nanoparticle to thecell or a cellular component (and/or its potential intracellular uptake)by at least one imaging technique. The imaging technique may be PET,SPECT, CT, MM, optical bioluminescence or fluorescence imaging, andcombinations thereof.

The location of the cellular component can be detected and determinedinside a metabolically active whole cell, in a whole cell lysate, in apermeabilized cell, in a fixed cell, or with a partially purified cellcomponent in a cell-free environment. The amount and the duration of thecontacting can depend, for example, on the diagnostic or therapeuticobjectives of the treatment method, such as fluorescent orantibody-mediated detection of upregulated signaling pathwayintermediates (i.e., Akt, NF-κB), disease states or conditions, thedelivery of a therapeutic agent, or both. The amount and the duration ofthe contacting can also depend on the relative concentration of thefluorescent nanoparticle to the target analyte, particle incubationtime, and the state of the cell for treatment.

The present invention further provides a method for targeting a tumorcell comprising administering to a cancer patient an effective amount ofa fluorescent silica-based nanoparticle comprising a silica-based corecomprising a fluorescent compound positioned within the silica-basedcore; a silica shell surrounding at least a portion of the core; anorganic polymer attached to the nanoparticle; a ligand attached to thenanoparticle and capable of binding a tumor marker; and at least onetherapeutic agent.

The nanoparticle may be radiolabeled. The nanoparticle may beradiolabelled using any suitable techniques. The radiolabelling may beautomated. In one embodiment, the nanoparticle is radiolabelled usingthe FASTlab radiochemistry synthesis platform (GE Healthcare). Thesynthesis parameters may be fine-tuned to achieve high reproducibility,radiochemical yield/purity, labeling efficiency, and relatively shortsynthesis times. New particle tracers may be accommodated on the sameFASTlab module.

The nanoparticle may be administered to the patient by, but notrestricted to, the following routes: oral, intravenous, nasal,subcutaneous, local, intramuscular or transdermal.

In certain embodiments, it may be desirable to use a mixture of two ormore types of fluorescent nanoparticles having different properties toevaluate different tissue types.

The methods and compositions of the invention can be used to help aphysician or surgeon to identify and characterize areas of disease, suchas cancers and inflammatory/infectious processes, including, but notrestricted to, cancers of the skin (melanoma), head & neck, prostate,brain, and bowels, to distinguish diseased and normal tissue, such asdetecting tumor margins that are difficult to detect using an ordinaryoperating microscope, e.g., in brain surgery, to help dictate atherapeutic or surgical intervention, e.g., by determining whether alesion is cancerous and should be removed or non-cancerous and leftalone, or in surgically staging a disease, e.g., intraoperative lymphnode staging, sentinel lymph node (SLN) mapping, e.g., nerve-sparingprocedures for preserving vital neural structures (intraparotid nerves).

The methods and compositions of the invention may be used in metastaticdisease detection, treatment response monitoring, SLN mapping/targeting,nerve sparing procedures, residual disease detection, targeted deliveryof therapeutics (combined diagnostic/therapeutic platform), localdelivery of non-targeted, drug-bearing nanoparticles (catheterdelivery), blood-brain barrier therapeutics, treatment ofinflammatory/ischemic diseases (i.e., brain, heart, urinary tract,bladder), combined treatment and sensing of disease (e.g., RatiometricpH sensing, oxygen sensing), etc.

The methods and compositions of the invention can also be used in thedetection, characterization and/or determination of the localization ofa disease, especially early disease, the severity of a disease or adisease-associated condition, the staging of a disease, and/ormonitoring a disease. The presence, absence, or level of an emittedsignal can be indicative of a disease state. The methods andcompositions of the invention can also be used to monitor and/or guidevarious therapeutic interventions, such as surgical and catheter-basedprocedures, and monitoring drug therapy, including cell based therapies.The methods of the invention can also be used in prognosis of a diseaseor disease condition. Cellular subpopulations residing within ormarginating the disease site, such as stem-like cells (“cancer stemcells”) and/or inflammatory/phagocytic cells may be identified andcharacterized using the methods and compositions of the invention. Withrespect to each of the foregoing, examples of such disease or diseaseconditions that can be detected or monitored (before, during or aftertherapy) include cancer (for example, melanoma, thyroid, colorectal,ovarian, lung, breast, prostate, cervical, skin, brain,gastrointestinal, mouth, kidney, esophageal, bone cancer), that can beused to identify subjects that have an increased susceptibility fordeveloping cancer and/or malignancies, i.e., they are predisposed todevelop cancer and/or malignancies, inflammation (for example,inflammatory conditions induced by the presence of cancerous lesions),cardiovascular disease (for example, atherosclerosis and inflammatoryconditions of blood vessels, ischemia, stroke, thrombosis), dermatologicdisease (for example, Kaposi's Sarcoma, psoriasis), ophthalmic disease(for example, macular degeneration, diabetic retinopathy), infectiousdisease (for example, bacterial, viral, fungal and parasitic infections,including Acquired Immunodeficiency Syndrome), immunologic disease (forexample, an autoimmune disorder, lymphoma, multiple sclerosis,rheumatoid arthritis, diabetes mellitus), central nervous system disease(for example, a neurodegenerative disease, such as Parkinson's diseaseor Alzheimer's disease), inherited diseases, metabolic diseases,environmental diseases (for example, lead, mercury and radioactivepoisoning, skin cancer), bone-related disease (for example,osteoporosis, primary and metastatic bone tumors, osteoarthritis) and aneurodegenerative disease.

The methods and compositions of the invention, therefore, can be used,for example, to determine the presence and/or localization of tumorand/or co-resident stem-like cells (“cancer stem cells”), the presenceand/or localization of inflammatory cells, including the presence ofactivated macrophages, for instance in peritumoral regions, the presenceand in localization of vascular disease including areas at risk foracute occlusion (i.e., vulnerable plaques) in coronary and peripheralarteries, regions of expanding aneurysms, unstable plaque in carotidarteries, and ischemic areas. The methods and compositions of theinvention can also be used in identification and evaluation of celldeath, injury, apoptosis, necrosis, hypoxia and angiogenesis.PCT/US2006/049222.

The following examples are presented for the purposes of illustrationonly and are not limiting the invention.

EXAMPLE 1 Preparation and Characterization of PEG-Coated Nanoparticles

Nanoparticles containing an NIR-emitting dye (Cy-5) were synthesized andfunctionalized by PEGylation according to well-established protocols asdisclosed in PCT/US2008/074894 and Stober et al. Controlled growth ofmonodispersed silica spheres in the micron size range. Colloid InterfaceSci. 1968; 26:62-69. Ohnishi et al. J. Mol. Imaging 2005, 4:172-181. Cy5malemide was reacted with a co-reactive organo silane compound,(3-Mercaptopropyl)tromethoxysilane to form a fluorescent silicaprecursor. This fluorescent silica precursor was co-condensed withtetraethylorthosilicate to form a fluorescent silica based core. APEG-silane compound, with methoxy-terminated poly(ethylene glycol)chains (PEG, ˜0.5 kDa) Methoxy(Polyethyleneoxy)Propyl]—Trimethoxysilane, was added to the fluorescent silica based coreto form a PEG coating on the core. PEG-coated nanoparticles weredialyzed to physiological saline (0.15M NaCl in H2O) through 3500 MWCOSnakeskin Dialysis Membranes and sterile-filtered. All samples wereoptical density-matched at their peak absorption wavelength (640 nm)prior to injection. Hydrodynamic size measurements were achieved byDynamic Light Scattering (DLS) and Fluorescence Correlation Spectroscopy(FCS). Briefly, particles dialyzed to water were measured on aBrookhaven Instruments Company 200SM static/DLS system using a HeNelaser (λ=632.8 nm). Due to overlap of the dye absorption with theexcitation source, 15-min integration times were used to achieveacceptable signal-to-noise ratios. For FCS, particles were dialyzed towater, diluted into 0.15M NaCl, and measured on a Zeiss LSM 510 Confocor2 FCS (HeNe 633 nm excitation). The instrument was calibrated for sizeprior to all measurements. Comparison of the relative brightness ofPEGylated nanoparticles with free dye was determined from FCS curves,measured as count rate per molecule/particle.

EXAMPLE 2 Renal Clearance of PEG Coated Nanoparticles

Fluorescent core-shell silica nanoparticles, having a hydrodynamicradius of about 3 nm, were synthesized. These nanoparticles were foundto be in the 6-10 nm diameter range, as shown by dynamic lightscattering (DLS) results (FIG. 1A). In vivo whole-body NIR fluorescenceimaging of bare (no PEG coat) silica nanoparticles, on the order of 6-nmand 3.3-nm, in nude mice showed considerable renal clearance 45 minpost-injection with a significant accumulation remaining in the liver(FIG. 1B). Eventual excretion into the enterohepatic circulationoccurred during the ensuing 24 h. On the basis of these results,particles were covalently coated with methoxy-terminated poly(ethyleneglycol) chains (PEG, ˜0.5 kDa), per protocols in PCT/US2008/074894, toprevent opsonization and further enhance particle clearance whilemaintaining a small hydrodynamic size. This treatment decreased liverretention and resulted in increased renal filtration into the bladder at45 min post-injection by NIR fluorescence imaging (FIG. 1C), withbladder fluorescence visible out to 24 h. The probes were welltolerated, with no adverse effects or animal deaths observed over thecourse of the study. Serial co-registered PET-CT imaging 24-hr afterinjection of ¹²⁴I-labeled PEG coated nanoparticles (FIG. 1D, upper row)demonstrated a small amount of residual bladder activity, as well asactivity overlying the liver/gastrointestinal tract (center), flanked byindependently acquired microCT and microPET scans. Serial microPETimages confirmed findings on NIR fluorescence imaging. The half-time ofblood residence of the ¹²⁴I-labeled PEGylated nanoparticles based ontime-dependent changes in blood activity over a 96-hour period was foundto be 7.3 hours. For the ¹²⁴I-labeled, RGD-bound nanoparticles, thehalf-time of blood residence was found to be 5.6 hours.

Based on these in vivo data, a more detailed biodistribution andclearance study of coated nanoparticles was undertaken on two sets ofPEGylated Cy5-containing particles to assess the effects of probe sizeon biodistribution. Nanoparticles with hydrodynamic diameters of3.3±0.06 and 6.0±0.1 nm, as measured by fluorescence correlationspectroscopy (FCS), were generated (FIG. 2A). Prior to in vivo studies,particle photophysical properties were investigated to establish theirperformance levels versus free dye. Despite the extremely small particlesize, silica-encapsulated dye molecules exhibited photophysicalenhancements over free dye that scaled with particle size, includingsignificant increases in brightness, as determined by absorption andemission spectroscopy (FIG. 2B) and FCS (FIG. 2C). Compared to the freedye, the 3.3 and 6.0 nm diameter nanoparticles exhibited 2- and 3-foldincreases in photobleaching half-life, respectively, when irradiatedwith a high power 635 nm laser (FIG. 2D). Thus, these nanoparticleprobes were found to be both brighter and more photostable than theirfree dye counterparts.

In addition to semiquantitative evaluation of in vivo nanoparticlebehavior from whole-body imaging, ex-vivo analysis of tissue homogenatesand fluids was performed using a fluorescence plate reader, whichallowed calibrated quantitation of variations observed in NIRfluorescence imaging. Samples were grouped as “retained” (liver, kidney,lung, spleen homogenates, and blood) and “excreted” (urine) sources ofparticle fluorescence, were background-corrected and were converted topercent of the initial dose (% ID) per animal based on calibrationcurves. Tissue analysis showed minimal particle retention in majororgans, with most of the fluorescence attributed to circulating blood(FIG. 3A). Net particle retention, calculated as the sum of the“retained” components, was fit with an exponential decay curve todetermine the kinetics of excretion (FIG. 3B). Larger particlesexhibited a longer tissue half-life (t_(1/2)(3.3 nm)=190 min,t_(1/2)(6.0 nm)=350 min) and greater initial organ retention. After 48h, the 6-nm particle exhibited minimal retention in the body(R_(total)(6.0 nm)=2.4±0.6% ID). Urine samples collected at the time ofsacrifice, in conjunction with serial dilution calibration data, wasused to estimate the total renal clearance based on a conservativeestimate of the average urine volume excreted per unit time. By thismethod, the % ID excreted over time for both particle sizes (FIG. 3C)was estimated.

EXAMPLE 3 Fluorescent Silica Nanoparticles Conjugated with α_(v)β₃Integrin-Targeting Peptide (Melanoma Model)

To synthesize a multimodal (optical-PET) nanoparticle with high affinityfor tumor marker α_(v)β₃ integrin, cyclic RGD pentapeptide (RGDYC (SEQID NO: 7)) was conjugated to the nanoparticle via a Cys-maleimidelinkage. The tyrosine linker, Y, was used to subsequently attach aradiolabel. Male athymic nude mice were injected subcutaneously intotheir flanks with C6 rat glioma cells. At ˜0.5 cm in diameter, mice wereIV-injected with either bare silica nanoparticles (FIG. 4A) orPEG-ylated RGD nanoparticles (FIG. 4B, ˜500 nm/kg). FIGS. 4A-4C show thein vivo biodistribution in non-tumor-bearing and tumor-bearing miceusing whole body optical imaging.

In vitro binding of targeted (RGD-bound) and non-targeted (PEG-coated)nanoparticles to α_(v)β₃-integrin-positive human melanoma cell lines(M21) was investigated as part of a dose response study using flowcytometry (FIGS. 5A, 5B). Particle binding/uptake was evaluated as afunction of time (FIG. 5A) and concentration (FIG. 5B).

EXAMPLE 4 Fluorescent Silica Nanoparticles Conjugated with α_(v)β₃Integrin-Targeting Peptide and Nodal Mapping (Melanoma Model)

We utilized a biocompatible material, silica, which has an architecturethat could be precisely tuned to particle sizes optimized for renalclearance. We attached small targeting peptides and a radioactive labelto the particle surface for serial PET imaging measurements in awell-characterized in vivo human melanoma model, and mapped draininglymph nodes and lymphatic channels using an encapsulated near infrared(NIR) dye and multi-scale optical fluorescence imaging methods. Ballouet al., Sentinel lymph node imaging using quantum dots in mouse tumormodels. Bioconjugate Chem. 18, 389-396 (2007). Kim et al., Near-infraredfluorescent type II quantum dots for sentinel lymph node mapping. Nat.Biotechnol. 22, 93-97 (2003). Tanaka et al, Image-guided oncologicsurgery using invisible light: completed pre-clinical development forsentinel lymph node mapping. J Surg Oncol. 13, 1671-1681 (2006).Toxicity testing was also performed and human normal-organ radiationdoses derived. Specifically, we synthesized about 7 nm diameter,near-infrared (NIR) dye-encapsulating core-shell silica nanoparticles,coated with PEG chains and surface-functionalized with a small number(about 6-7) of targeting peptides and radiolabels.

We demonstrate that these probes simultaneously are non-toxic, exhibithigh-affinity/avidity binding, efficient excretion, and significantdifferential uptake and contrast between tumor and normal tissues usingmultimodal molecular imaging approaches. The sensitive detection,localization, and interrogation of lymph nodes and lymphatic channels,enabled by the NIR dye fluorescence, highlights the distinct potentialadvantage of this multimodal platform for detecting and stagingmetastatic disease in the clinical setting, while extending the lowerrange of nodal sizes that can be detected.

Materials and Methods

Synthesis of cRGDY-PEG-Nanoparticles and PEG-Nanoparticles

Particles were prepared by a modified Stober-type silica condensation asdescribed previously. Wiesner et al., PEG-coated Core-shell SilicaNanoparticles and Mathods of Manufactire and Use, PCT/US2008/74894.Larson, et al., Silica nanoparticle architecture determines radiativeproperties of encapsulated chromophores. Chem. Mater. 20, 2677-2684(2008). Bogush, et al., Preparation of Monodisperse Silica Particles:Control of Size and Mass Fraction. J. Non-Cryst. Solids, 104, 95-106(1988). Sadasivan, et al., Alcoholic Solvent Effect on SilicaSynthesis—NMR and DLS Investigation. J. Sol-Gel Sci. Technol. 12, 5-14(1998). Herz, et al., Large Stokes-Shift Fluorescent SilicaNanoparticles with Enhanced Emission over Free Dye for Single ExcitationMultiplexing. Macromol Rapid Commun. 30, 1907-1910 (2009). Tyrosineresidues were conjugated to PEG chains for attachment of radioiodine orstable iodine moieties. Hermanson, Bioconjugate Techniques, (AcademicPress, London, ed. 2, 2008). All samples were optical density-matched attheir peak absorption wavelength (640 nm) prior to radiolabeling. cRGDpeptides were attached to functionalized PEG chains via acysteine-maleimide linkage, and the number of cRGD peptides bound to theparticle was estimated using FCS-based measurements of absolute particleconcentrations and the starting concentration of the reagents for cRGDpeptide.

Hydrodynamic Size and Relative Brightness Comparison Measurements byFluorescence Correlation Spectroscopy (FCS)

Particles dialyzed to water were diluted into physiological saline (0.15M NaCl in H2O) and measured on a Zeiss LSM 510 Confocor 2 FCS using HeNe633-nm excitation. The instrument was calibrated for size prior to allmeasurements. Diffusion time differences were used to evaluatevariations in the hydrodynamic sizes of the dye and particle species.Relative brightness comparisons of the free dye and the PEG- and theRGDY-PEG nanoparticles were performed using count rates permolecule/particle.

Radiolabeling of C Dot Conjugates

Radiolabeling of the cRGDY-PEG and PEG-nanoparticles was performed usingthe IODOGEN method (Pierce, Rockford, Ill.). Piatyszek, et al., Iodo-genmediated radioiodination of nucleic acids. J. Anal. Biochem. 172,356-359 (1988). Activities were measured by gamma (γ)-counting andfluorescence measured using a Varian fluorometer (excitation 650nm/emission 680).

Cells and Cell Culture

Human melanoma M21 and M21 variant (M21-L, α_(v) negative) cell lineswere maintained in RPMI 1640 media/10% fetal BSA, 2 mM L-glutaminepenicillin, and streptomycin (Core Media Preparation Facility, MemorialSloan Kettering Cancer Center, New York). Human umbilical venous cordendothelial cells (HUVECs) were cultured in M199 media/10% fetal bovineserum, 20 μg/ml endothelial cell growth factor, 50 μg/ml heparin,penicillin and streptomycin.

In Vitro Cell-Binding and Molecular Specificity of¹²⁴I-cRGD-PEG-Nanoparticles

To assay particle binding and specificity for M21 cells, 24-well plateswere coated with 10 μg/ml collagen type I (BD Biosciences, Bedford,Mass.) in phosphate buffered saline (PBS) and incubated (37° C., 30min). M21 cells (3.0-4.0×105 cells/well) were grown to confluency andwashed with RPMI 1640 media/0.5% bovine serum albumin (BSA).¹²⁴I-cRGD-PEG-nanoparticles (0-4.0 ng/ml) were added to wells and cellsincubated (25° C., 4 hours), washed with RPMI 1640 media/0.5% BSA, anddissolved in 0.2 M NaOH. Radioactivity was assayed using a 1480Automatic Gamma Counter (Perkin Elmer) calibrated for iodine-124.Nonspecific binding was determined in the presence of a 1000-fold excessof cRGD (Peptides International, Louisville, Ky.). Scatchard plots ofthe binding data were generated and analyzed using linear regressionanalyses (Microsoft Excel 2007) to derive receptor-binding parameters(Kd, Bmax, IC50).

In Vitro Cell-Binding Studies Using Optical Detection Methods

Maximum differential binding of cRGDY-PEG-nanoparticles andPEG-nanoparticles to M21 cells was determined for a range of incubationtimes and particle concentrations using flow cytometry, with optimumvalues used in competitive binding and specificity studies. Cells(3.0×10⁵ cells/well) were washed with RPMI 1640 media/0.5% BSA, detachedusing 0.25% trypsin/EDTA, and pelleted in a microcentrifuge tube (5 minat 1400 rpm, 25° C.). Pellets were resuspended in BD FACSFlow solution(BD Biosciences, San Jose, Calif.) and analyzed in the Cy5 channel todetermine the percentage of particle-bound probe (FACSCalibur, BectonDickinson, Mountain View, Calif.). Competitive binding studies wereadditionally performed following incubation of cRGDY-PEG-nanoparticles(2.5 ng/ml) with M21, M21L, and HUVEC cells in the presence of excesscRGD and/or mouse monoclonal anti-human integrin α_(v)β₃fluorescein-conjugated antibody (Millipore, Temecula, Calif.) andanalyzed by flow cytometry. To assess potency of the RGDY-PEGnanoparticles relative to the cRGD peptide, anti-adhesion assays wereperformed. Ninety-six-well microtiter plates were coated withvitronectin in PBS (5 μm/ml), followed by 200 μl of RPMI/0.5% BSA (1 h,37° C.). Cells (3×10⁴/100 μl/well) were incubated in quadruplicate (30min, 25° C.) with various concentrations of cRGDY-PEG-nanoparticles orcRGD peptide in RPMI/0.1% BSA, and added to vitronectin-coated plates(30 min, 37° C.). Wells were gently rinsed with RPMI/0.1% BSA to removenon-adherent cells; adherent cells were fixed with 4% PFA (20 min, 25°C.) and stained with methylene blue (1 h, 37° C.) for determination ofoptical densities, measured using a Tecan Safire plate reader (λex=650nm, λem=680 nm, 12 nm bandwidth). The multivalent enhancement factor wascomputed as the ratio of the cRGD peptide to cRGDY-PEG-dot IC50 values.Montet, et al., Multivalent effects of RGD peptides obtained bynanoparticle display. J Med Chem. 49, 6087-6093 (2006).

Animal Models and Tumor Innoculation

All animal experiments were done in accordance with protocols approvedby the Institutional Animal Care and Use Committee of MemorialSloan-Kettering Cancer Center and followed National Institutes of Healthguidelines for animal welfare. Male athymic nu/nu mice (6-8 weeks old,Taconic Farms Inc, Hudson, N.Y.) were provided with water containingpotassium iodide solution to block uptake by the thyroid gland of anyfree radioiodine in vivo, and maintained on a Harlan Teklad Global Diet2016, ad libitum, as detailed elsewhere 10. To generate M21 or M21Lxenografts, equal volumes of cells (˜5×10⁶/100 μl) and matrigel wereco-injected subcutaneously into the hindleg in different mice. Tumorsizes were regularly measured with calipers, yielding average tumorvolumes of 200 mm³.

In Vivo Pharmacokinetic and Residence Half-Time (T_(1/2)) Measurements

Time-dependent activity concentrations (% ID/g), corrected forradioactive decay to the time of injection, were measured by sacrificinggroups of mice at specified times following i.v. injection of¹²⁴I-cRGDY-PEG-nanoparticles or ¹²⁴I-PEG-nanoparticles (˜20 μCi/mouse)and harvesting, weighing, and counting blood, tumor, and organs in ascintillation γ-counter calibrated for ¹²⁴I. The resulting time-activityconcentration data for each tissue were fit to a decreasingmonoexponential function to determine the values of T_(1/2) and A, thetissue/organ residence half time and zero-time intercept, respectively,of the function.

The fraction of particles excreted in the urine over time was estimatedusing previously described methods. Burns, et al., Fluorescent SilicaNanoparticles with Efficient Urinary Excretion for Nanomedicine, NanoLetters 9, 442-8 (2009). Briefly, mice were injected i.v. with either200 μl unlabeled cRGDY-PEG-nanoparticles or PEG-nanoparticles, and urinesamples collected over a 168-hr period (n=3 mice per time point).Particle concentrations at each time point were determined usingfluorometric analyses and a serial dilution calibration curve generatedfrom background-corrected fluorescence signal measurements of urinesamples mixed with known particle concentrations (% ID). Concentrationvalues, along with estimates of average daily mouse urine volumes, werethen used to compute the cumulative % ID/g urine excreted over time. Toassess cumulative fecal excretion, metabolic cages were used to collectfeces over a similar time interval after i.v. injection of 200 μl¹²⁴I-cRGDY-PEG-nanoparticles (n=4 mice per time point). Specimenactivities were measured using a γ-counter calibrated for ¹²⁴I.

Dosimetry

Time-activity functions derived for each tissue were analyticallyintegrated (with inclusion of the effect of radioactive decay) to yieldthe corresponding cumulative activity (i.e. the total number ofradioactive decays). ¹²⁴I mouse organ absorbed doses were thencalculated by multiplying the cumulative activity by the ¹²⁴Iequilibrium dose constant for non-penetrating radiations (positrons),assuming complete local absorption of such radiations and ignoring thecontribution of penetrating radiations (i.e., γ-rays). Eckerman, et al.,Radionuclide Data and Decay Schemes, 2nd ed. Reston, Va.: Society ofNuclear Medicine; 1989. The mouse normal-organ cumulated activities wereconverted to human normal-organ cumulated activities by adjustment forthe differences in total-body and organ masses between mice and humans(70-kg Standard Man). Cristy, et al., Specific absorbed fractions ofenergy at various ages from internal photon sources (I-VII). Oak RidgeNational Laboratory Report ORNL/TM-8381/V1-7. Springfield, Va.: NationalTechnical Information Service, Dept of Commerce; 1987. The humannormal-organ cumulated activities calculated were entered into theOLINDA dosimetry computer program to calculate, using the formalism ofthe Medical Internal Dosimetry (MIRD) Committee of the Society ofNuclear Medicine, the Standard-Man organ absorbed doses. Loevinger, etal., MIRD Primer for Absorbed Dose Calculations (Society of NuclearMedicine, New York, 1991). Stabin, et al., OLINDA/EXM: thesecond-generation personal computer software for internal doseassessment in nuclear medicine. J Nucl Med. 46, 1023-1027 (2005).

Acute Toxicity Studies and Histopathology

Acute toxicity testing was performed in six groups of male and femaleB6D2F1 mice (7 wks old, Jackson Laboratories, Bar Harbor, Me.). Thetreatment group (n=6 males, n=6 females) received unlabeled targetedprobe (¹²⁷I-RGDY-PEG-nanoparticles) and the control group (n=6 males,n=6 females) unlabeled iodinated PEG-nanoparticles (vehicle,¹²⁷I-RGDY-PEG-nanoparticles) in a single i.v. injection (200 μl).Untreated controls (n=2 males, n=2 females) were additionally tested.Mice were observed daily over 14 days p.i. for signs ofmorbidity/mortality and weight changes, and gross necropsy,histopathology, and blood sampling for hematology and serum chemistryevaluation was performed at 7- and 14-days p.i (FIGS. 10A-10B and Table3).

Serial PET Imaging of Tumor-Specific Targeting

Imaging was performed using a dedicated small-animal PET scanner (Focus120 microPET; Concorde Microsystems, Nashville, Tenn.). Mice bearing M21or M21L hindleg tumors were maintained under 2% isoflurane anesthesia inoxygen at 2 L/min during the entire scanning period. One-hour list-modeacquisitions were initiated at the time of i.v. injection of 200 μCi of¹²⁴I-cRGDY-PEG-nanoparticles or ¹²⁴I-PEG-nanoparticles in all mice,followed by serial 30 min static images over a 96-hour interval. Imagedata were corrected for non-uniformity of the scanner response, deadtime count losses, random counts, and physical decay to the time ofinjection. Voxel count rates in the reconstructed images were convertedto activity concentrations (% ID/g) by use of a measured systemcalibration factor. Three-dimensional region-of-interest (ROI) analysisof the reconstructed images was performed by use of ASIPro software(Concorde Microsystems, Nashville, Tenn.) to determine the mean,maximum, and SD of probe uptake in the tumors. Tumor-to-muscle activityconcentration ratios were derived by dividing the image-derived tumor %ID/g values by the γ-counter muscle % ID/g values.

Nodal Mapping Using Combined NIR Fluorescence Imaging and Microscopy

Nude mice bearing hindleg tumors were injected by 4-quadrant,peritumoral administration using equal volumes of a 50-μ1 cRGDY-PEG-dotsample and allowed to perambulate freely. Following a 30 min to 1-hrinterval, mice were anesthetized with a 2% isofluorine/98% oxygenmixture, and a superficial paramidline incision was made verticallyalong the ventral aspect of the mouse to surgically expose the regionfrom the hindlimb to the axilla ipsilateral to the tumor. In situoptical imaging of locoregional nodes (i.e., inguinal, axillary) anddraining lymphatics (including axillary region) was performed using amacroscopic fluorescence microscope fitted with 650±20 nm NIR excitationand 710-nm long-pass emission filters. Whole-body optical images(Cambridge Research Instruments Maestro imager) were additionallyacquired and spectrally deconvolved as reported previously. Burns, etal., Fluorescent Silica Nanoparticles with Efficient Urinary Excretionfor Nanomedicine, Nano Letters 9, 442-8 (2009).

Statistical Analysis

Statistical analyses comparing groups of tumor mice receivingtargeted/non-targeted probes or bearing M21/M21L tumors, were performedusing a one-tail Mann-Whitney U test, with P<0.05 consideredstatistically significant. For biodistribution studies, thetissue-specific mean % ID/g values of ¹²⁴I-cRGDY-PEG- (n=7 mice) and¹²⁴I-PEG-nanoparticles (control, n=5 mice) were compared at each timepoint, with statistically significant differences in tracer activitiesobserved in blood, tumor, and major organs at 4 and 96 hrs p.i., as wellas at 24 hrs p.i. for tumor and other tissues (Table 1). For tumortargeting studies, differences in mean % ID/g values between M21 (n=7)and M21L tumor mice (n=5), as well as mice receiving control probes(n=5), were found to be maximal at 4 hrs p.i. (p=0.0015 for bothcontrols), remaining significantly elevated at 24 hrs (p=0.0015 andp=0.004, respectively), 48 hrs (p=0.001 and p=0.003, respectively), 72hrs (p=0.015 and 0.005, respectively), and 96 hrs (p=0.005 forM21-M21L). Tumor-to-muscle ratios for ¹²⁴I-cRGDY-PEG-nanoparticles (n=7)versus ¹²⁴I-PEG-nanoparticles (n=5) were found to be statisticallysignificant at 24 hrs p.i. (p=0.001) and 72 hrs p.i. (p=0.006), but notat 4 hrs p.i. (p=0.35). Goodness of fit values (R2), along with theirassociated p values, were determined for the urine calibration curve(R2=0.973, p=0.01), as well as for the urine (R2>0.95, p=0.047) andfecal (R2>0.995, p<0.002) cumulative % ID excretion curves usingnon-linear regression analyses (SigmaPlot, Systat, v. 11.0).

Results

Nanoparticle Design and Characterization

Cy5 dye encapsulating core-shell silica nanoparticles (emissionmaxima >650 nm), coated with methoxy-terminated polyethylene glycol(PEG) chains (PEG ˜0.5 kDa), were prepared according to previouslypublished protocols. Burns, et al., Fluorescent Silica Nanoparticleswith Efficient Urinary Excretion for Nanomedicine, Nano Letters, 9,442-8 (2009). Ow, et al., Bright and stable core-shell fluorescentsilica nanoparticles. Nano Lett. 5, 113-117 (2005). The neutral PEGcoating prevented non-specific uptake by the reticuloendothelial system(opsonization). The use of bifunctional PEGs enabled attachment of smallnumbers (˜6-7 per particle) of α_(v)β₃ integrin-targeting cyclicarginine-glycine-aspartic acid (cRGDY (SEQ ID NO: 1)) peptide ligands tomaintain a small hydrodynamic size facilitating efficient renalclearance. Peptide ligands were additionally labeled with ¹²⁴I throughthe use of a tyrosine linker to provide a signal which can bequantitatively imaged in three dimensions by PET (¹²⁴I-cRGDY-PEG-dots,FIG. 6A); an important practical advantage of relatively long-lived ¹²⁴I(physical half-life: 4.2 d) is that sufficient signal persists longenough to allow radiodetection up to at least several dayspostadministration, when background activity has largely cleared andtumor-to-background contrast is maximized. Purity of the radiolabeledtargeted nanoparticle was >95% by radio thin layer chromatography.Stability of the non-radiolabeled targeted nanoparticle is about 1 yearby FCS measurements. Particle is excreted intact in the urine by FCSanalyses. As used herein, “dot” and “nanoparticle” are usedinterchangeably. A PEG-coated particle containing a tyrosine residue for¹²⁴I labeling served as the control probe (¹²⁴I-PEG-dots). Purificationof the radiolabeled samples by size exclusion chromatography (FIG. 7)resulted in radiochemical yields of >95%. Hydrodynamic diameters of ˜7nm i.d. were measured for non-radioactive cRGDY-PEG-dots and PEG-dots byfluorescence correlation spectroscopy (FCS) (FIGS. 6B and 6C). Therelative brightness of the cRGDY-PEG-dots was determined, on average, tobe 200% greater than that of the free dye (FIG. 6C), consistent withearlier results. Burns, et al., Fluorescent Silica Nanoparticles withEfficient Urinary Excretion for Nanomedicine, Nano Letters, 9, 442-8(2009). Larson, et al., Silica nanoparticle architecture determinesradiative properties of encapsulated chromophores. Chem. Mater. 20,2677-2684 (2008). Based on these physicochemical properties, weanticipated achieving a favorable balance between selective tumor uptakeand retention versus renal clearance of the targeted particle, thusmaximizing target-tissue localization while minimizing normal-tissuetoxicity and radiation doses.

In Vitro Receptor Binding Studies

To examine in vitro binding affinity and specificity of¹²⁴I-cRGDY-PEG-dots and ¹²⁴I-PEGdots to tumor and vascular endothelialsurfaces, α_(v)β₃ integrin-overexpressing (M21) and nonexpressing (M21L)melanoma and human umbilical vein endothelial (HUVECs) cell lines wereused. Highly specific linear and saturable binding of the cRGDY-PEG-dotswas observed over a range of particle concentrations (0 to 8 ng/ml) andincubation times (up to 5-hrs), with maximum differential binding at4-hr and ˜2.0 ng/ml particle concentration (data not shown) using flowcytometry. Receptor-binding specificity of ¹²⁴I-cRGDY-PEG dots wastested using γ-counting methods after initially incubating M21 cellswith excess non-radiolabeled cRGD and then adding various concentrationsof the radiolabeled targeted probe (FIG. 8A). Scatchard analysis of thebinding data yielded a dissociation equilibrium constant, Kd, of 0.51 nM(FIG. 8A, inset) and receptor concentration, Bmax, of 2.5 pM. Based onthe Bmax value, the α_(v)β₃ integrin receptor density was estimated tobe 1.0×10⁴ per M21 cell, in reasonable agreement with the previouslypublished estimate of 5.6×10⁴ for this cell line. Cressman, et al.,Binding and uptake of RGD-containing ligands to cellular α_(v)β₃integrins. Int J Pept Res Ther. 15, 49-59 (2009). Incremental increasesin integrin-specific M21 cellular uptake were also observed over atemperature range of 4 to 37° C., suggesting that receptor-mediatedcellular internalization contributed to overall uptake (data not shown).Additional competitive binding studies using the targeted probe showedcomplete blocking of receptor-mediated binding with anti-α_(v)β₃integrin antibody (FIG. 8B) by flow cytometry. No significant reductionwas seen in the magnitude of receptor binding (˜10% of M21) with M21Lcells (FIG. 8C) using either excess cRGDY (SEQ ID NO: 1) or anti-α_(v)β₃integrin antibody. These results were confirmed by additional γ-countingstudies, and a 50% binding inhibition concentration, IC50, of 1.2 nM wasdetermined for the ¹²⁴I-cRGDY-PEG-dot. An associated multivalentenhancement factor of greater than 2.0 was found for the cRGDY-PEG-dotrelative to the monomeric cRGD peptide using an anti-adhesion assay andM21 cells (data not shown). Montet, et al., Multivalent effects of RGDpeptides obtained by nanoparticle display. J Med Chem. 49, 6087-6093(2006). Li, et al., ⁶⁴Cu-labeled tetrameric and octomeric RGD peptidesfor small-animal PET of tumor α_(v)β₃ integrin expression. J. Nucl Med.48, 1162-1171 (2007). Similar to M21 cells, excess antibody effectivelyblocked cRGDY-PEG-dot receptor binding to HUVEC cells by flow cytometry(FIG. 8D).

Biodistribution and Clearance Studies

The time-dependent biodistribution, as well as renal and hepatobiliaryclearance were evaluated by intravenously administering tracer doses(˜0.2 nanomoles) of ¹²⁴I-cRGDY-PEGdots and ¹²⁴I-PEG-dots to M21 tumorxenograft mouse models (FIGS. 9A-9D). Although tissueactivity-concentrations (percent of the injected dose per gram (% ID/g))for the targeted probe were measured over a 196-hr post-injection (p.i.)time interval, comparison of the ¹²⁴I-cRGDY-PEGdot (FIG. 9A) and¹²⁴I-PEG-dot tracers (FIG. 9B) was restricted to a 96-hr window, as datafor the latter was not acquired at 1 week. Statistically significant(p<0.05) differences in tracer activities were observed for blood,tumor, and major organs at 4 and 96 hrs p.i., as well as at 24 hrs p.i.for the tumor and several other tissues (Table 1). The targeted probewas almost entirely eliminated from the carcass at 1 week p.i (˜3% ID).The residence half times (T_(1/2)) for blood, tumor, and major organsfor these tracers are shown in Table 2 (columns 2 and 5). Arepresentative data set (blood residence) is shown in the inset of FIG.9A. A relatively long blood T_(1/2) value of 7.3±1.2 hrs was determinedfor the ¹²⁴I-PEG-dot. Upon attachment of the cRGDY peptide (SEQ IDNO: 1) to synthesize the ¹²⁴I-cRGDY-PEG-dot, the T_(1/2) value decreasedslightly to 5.6±0.15 hrs, but was accompanied by greater probebioavailability (Table 2, column 3). The tumor T_(1/2) value for the¹²⁴I-cRGDY-PEG-dot was found to be about 13 times greater than that forblood, versus only a 5-fold difference for the ¹²⁴I-PEG-dot (Table 2,columns 2 and 5).

TABLE 1 Biodistribution study p-values comparing ¹²⁴I-cRGDY-PEG-and¹²⁴I-PEG-dots^(†) Post-injection times (hours) Tissue 4 24 96 Blood0.001 0.113 0.010 Tumor 0.045 0.012 0.001 Heart 0.019 0.231 0.001 Lungs— 0.039 0.006 Liver 0.001 0.033 0.028 Spleen 0.001 0.208 0.001 SmallIntestine 0.001 0.046 0.002 Large Intestine 0.001 0.137 0.003 Kidneys —0.356 0.001 Muscle 0.001 0.007 0.001 Brain 0.001 0.074 0.001

TABLE 2 Mouse Human^(†) ¹²⁴I-RGDY-PEG ¹²⁴I-PEG ¹²⁴I RGDY-PEG ¹²⁴I-PEGT_(1/2) A Absorbed Dose T_(1/2) A Absorbed Dose Absorbed Dose TargetOrgan (h) (% ID/g) (rad/mCi) (h) (% ID/g) (rad/mCi) (rad/mCi) Blood 5.918.8 626 7.3 4.7 189 (see red marrow below) Heart 6.8 7.0 266 34.1 0.8120 0.307 (Wall) 0.087 Lungs 8.5 5.7 267 37.7 3.0 498 0.298 0.263 Liver65.9 3.9 935 52.5 1.4 294 0.486 0.234 Spleen 42.3 45.6 1071 27.4 45.7410 3.20 0.254 195. 286 Small Intestine 30.3 1.8 251 13.2 0.9 61 0.3040.115 Large Intestine 23.9 2.0 228 49.2 0.5 99 0.427 (U) 0.209 0.724 (L)0.416 Kidneys 66.0 3.0 712 33.0 2.0 388 2.50 0.320 Muscle 27.7 0.8 10547.1 0.2 38 0.227 0.060 Brain 13.9 0.4 29 8.5 0.2 8 0.187 0.149^(§)Tumor 73.5 1.5 380 37.0 0.9 146 n/a n/a ^(ζ)Bone (See osteogeniccells) Adrenals 0.400 0.083 Breasts 0.141 0.042 Gallbladder Wall 0.2890.097 Stomach Wall 0.265 0.065 Ovaries 0.303 0.124 Pancreas 0.389 0.081Red Marrow 1.07 0.084 Osteogenic Cells 0.203 0.127 Skin 0.158 0.038Testes 0.186 0.073 Thymus 0.173 0.052 Thyroid 0.188 0.043 UrinaryBladder Wall 2.01 1.65 Uterus 0.333 0.171 Total Body 0.034 0.075Effective Dose Equivalent (rem/mCi) 0.863 0.256 Effective Dose (rem/mCi)0.599 0.232 ^(†)70-kg Standard Man, U (upper), L (lower), ^(§)mousemelanoma model, ^(ζ)bone activity much lower than other tissues (notreported)

By appropriate mass-adjusted translation of the foregoingbiodistribution data to man, human normal-organ radiation doses werederived and found to be comparable to those of other commonly useddiagnostic radiotracers (Table 2, columns 8, 9). Along with the findingthat the targeted probe was non-toxic and resulted in no tissue-specificpathologic effects (i.e., no acute toxicity) (FIGS. 10A-10B and Table3), first-in-man targeted and nontargeted molecular imaging applicationswith these agents are planned.

TABLE 3 Organ Histopathology for ¹²⁷I-RGDY-PEG-DOTS vs ¹²⁷I-PEG-DOTSTreatment UNTREATED ¹²⁷I-PEG-DOTS ¹²⁷I-RGDY-PEG-DOTS Sex M F M M F F M MF F Heart N N N N N N N N N N Thymus N N N N N N N N N N Trachea N N N NN N N N N N Lungs N N N N N N N N N N Kidneys N N N N N N N N N N LiverN N N N N N N N N N Random cellular clusters 1 Gall bladder NP N N N NNP N NP N N Pancreas N N N N N N N N N N Chronic lymph 2F N N N N SpleenN N N N N N N N N N Salivary gland N N N N N N N N N N Esophagus N N N NN N N N N N Stomach N N N N N N N N N N Small intestine N N N N N N N NN N Follic lymph. 1 Hyperplasia MF Large intestine N N N N N N N N N NMesenteric lymph node N N N N N N N N N NP Submandibular lymph NP NP NNP N N N N N N Adrenals N N N N N N N N N N Thyroid N N N N N N N N N NTestes N U N N U U N N U U Epididymides N U N N U U N N U U Seminalvesicles N U N N U U N N U U Coagulating glands N U N N U U N N U UProstate N U N N U U N N U U Ovary U N U U N N U U N N Uterus U N U U NN U U N N Cervix U N U U N N U U N N Mammary gland NP NP N NP N N NP NPN NP Urinary bladder N N N N N N N N N N Bones, joint N N N N N N N N NN Bone marrow N N N N N N N N N N Spinal cord N N N N N N N N N N BrainN N N N N N N N N N Pituitary N NP N N N N N N N N Skin N N N N N N N NN N Subcut. Inflammation 1 Skeletal muscle N N N N N N N N N NPeripheral nerves N N N N N N N N N N N: normal, U: unavailable, NP: notpresent, 1: minimal, 2: mild F: focal, MF: multifocal

In another study to confirm that ¹²⁷I-RGD-PEG dots are non-toxic afterintravenous administration in mice, formal single dose toxicity testingwas performed over the course of 2 weeks using ¹²⁷I-RGD-PEG dots atabout 100 times of the human dose equivalent. ¹²⁷I-PEG dots served asthe control particle. In summary, the procedure was as follows.Twenty-eight, 8 week old B6D2F1 mice were used in the acute toxicitystudy and were divided into a treatment and control group. The treatmentgroup (n=6 males+6 females) received one dose ¹²⁷I-PEGylated RGD silicananoparticles at a dose of 1×10⁻⁹ moles/mouse intravenously, and thecontrol group (n=6 males+6 females) received the same amount of vehicle.Two mice/group (one male and one female/group) were sacrificed on day 7post dose and clinical chemistry, hematology and tissue specifichistopathology were done at autopsy. All remaining animals (n=5 males+5females/group) were observed for 14 days following treatment. Fouruntreated mice (two males and two females) were used as reference. Theconclusion of the studies was that no adverse events were observedduring dosing or the following 14-days observation period. No mortalityor morbidity was observed. Clinical observations included the absence ofthe following: anemia, weight loss, agitation, increased respiration, GIdisturbance, abnormal behavior, neurological dysfunction, abnormalitiesin hematology, abnormalities in clinical chemistries, or drug-relatedlesions in terms of organ pathology. Thus, a single injection of¹²⁷I-PEGylated RGD silica nanoparticles at 1×10⁻⁹ moles/mouse, a doseequivalent to an excess of 100 times the PEGylated RGD silicananoparticles dose required for Phase 0 imaging studies, is safe andnontoxic in B6D2F1 mice.

Efficient renal excretion was found for the ˜7-nm diameter targeted andnon-targeted probes over a 168-hr time period by fluorometric analysesof urine samples. Fluorescence signals were background-corrected andconverted to particle concentrations (% ID/μ1) based on a serialdilution calibration scheme (FIG. 9C, inset; Table 4, column 2). Burns,et al., Fluorescent Silica Nanoparticles with Efficient UrinaryExcretion for Nanomedicine, Nano Letters, 9, 442-8 (2009). Concentrationvalues, along with age-dependent conservative estimates of the averageurine excretion rate, permitted the cumulative % ID excreted to becomputed (Table 4, column 4). Drickamer, Rates of urine excretion byhouse mouse (mus domesticus): differences by age, sex, social status,and reproductive condition. J. Chem. Ecol. 21, 1481-1493 (1995). Nearlyhalf of the injected dose (about 43% ID) was observed to be excretedover the first 24 hrs p.i. and ˜72% ID by 96 hrs, FIG. 9C), suggestingthat the bulk of excretion has occurred in the first day p.i. Nosignificant particle fluorescence in urine could be detected 168 hrsp.i. Fecal excretion profiles of the ¹²⁴I-cRGDY-PEG-dot indicated that,on average, 7% ID and 15% ID of the injected dose was eliminated over 24and 96 hrs, respectively (FIG. 9D). FCS analysis of urine samplesobtained at multiple time points after injection of the targeted proberevealed that the particle was excreted intact and without release ofthe encapsulated dye (data not shown).

TABLE 4 Urine Concentration and Cumulative Excretion Data ComputedConcentration Avg. Urine Cumulative Time (hr) (% ID/ul) Volume (μl) % IDExcreted 7.0 mm 0 0.0 0.0 0 RGDY-PEG 1 0.292 41.6 6.07 dot 4 0.026 166.726.1 24 0.016 1000. 43.4 96 0.004 3974. 72.2Serial Whole Body PET Studies

PET imaging of integrin expression in M21 and M21L subcutaneous hindlegxenograft mouse models was performed at multiple time points p.i.following i.v. injection of ¹²⁴I-cRGDY-PEG-dots or ¹²⁴I-PEG-dots(control). Representative whole-body coronal microPET images at 4 hrs(left: M21 tumor; middle: M21L tumor) and 24 hrs (right: M21 tumor) p.i.are shown in FIG. 11A. The specific targeting of the α_(v)β₃integrin-overexpressing M21 tumor is clearly visible from these images.Average tumor % ID/g and standard deviations are shown for groups of M21(n=7) and M21L (control) tumors (n=5) receiving the targeted¹²⁴I-cRGDY-PEG-dots, as well as for M21 tumor mice (n=5) receivingnon-targeted ¹²⁴I-PEG-dot tracer (FIG. 11B). At the time of maximumtumor uptake (˜4 hrs p.i.), three-fold activity-concentration increases(in % ID/g) were seen in the M21 tumors over the controls. Differenceswere statistically significant at all time points p.i. (p<0.05) exceptat 1 hr (p=0.27).

Image-derived tumor-to-muscle uptake (% ID/g) ratios for the¹²⁴I-cRGDY-PEG-dots revealed enhanced tumor contrast at later times(˜24-72 hrs p.i.), while that for ¹²⁴I-PEG-dots declined (FIG. 11C).This finding suggested that ¹²⁴I-cRGDY-PEG-dots were, in fact,tumor-selective, which became more apparent as the blood activity wascleared during the initial 24-hr period (compare FIG. 11C with inset ofFIG. 9A). A statistically significant correlation was found betweenPET-derived tumor tissue % ID/g values for both ¹²⁴I-cRGDY-PEG-dots and¹²⁴I-PEGdots, and the corresponding ex-vivo γ-counted tumor % ID/gvalues (correlation coefficient r=0.94, P<0.0016; FIG. 11D), confirmingthe accuracy of PET for non-invasively deriving quantitativebiodistribution data.

In Vivo NIR Fluorescence Imaging and Microscopy

We performed in vivo fluorescence imaging studies using our small,targeted nanoparticles for mapping local/regional nodes and lymphaticchannels, thus overcoming the foregoing limitation. Importantly, themultimodal nature and small size of our targeted particle probe can beexploited to visualize a range of nodal sizes and lymphatic branches inour melanoma model following 4-quadrant, peritumoral administration,simulating intraoperative human sentinel lymph node mapping procedures.Initially, serial NIR fluorescence microscopy was performed in intactmice over a 4-hr time period using either the targeted or non-targetedparticle probes. Peritumoral administration of the targeted proberevealed drainage into and persistent visualization of adjacent inguinaland popliteal nodes over this interval, with smaller and/or more distantnodes and lymphatics more difficult to visualize. By contrast, thenon-targeted probe yielded shorter-term (˜1 hr) visualization of localnodes with progressively weaker fluorescence signal observed (data notshown). Upon surgical exposure, this observation was found to be theresult of more rapid particle diffusion from the tumor site, as comparedwith the extended retention observed with the targeted probe.

We next performed representative lymph node mapping over multiplespatial scales using live-animal whole-body optical imaging (FIG. 12A)and NIR fluorescence microscopy techniques (FIG. 12B) to visualizelymphatic drainage from the peritumoral region to the inguinal andaxillary nodes in surgically exposed living animals. In addition,higher-resolution fluorescence images (FIG. 12B, lower row) permittedmore detailed intranodal architecture to be visualized, including highendothelial venules, which facilitate passage of circulating naïvelymphocytes into the node, and which may have important implications fornodal staging and the ability to detect micrometastases at earlierstages of disease. Smaller, less intense lymphatic branches were alsovisualized by fluorescence microscopy in the axillary region (data notshown). Thus, the small size of the targeted probe not only permits thefirst draining (or sentinel node), proximal to the tumor to bevisualized, but also enables visualization of more distant nodes and ofthe pattern of lymphatic drainage to be visualized.

Discussion

We report on non-toxic, high-affinity, and efficiently cleared silicananoparticles for tumor-selective targeting and nodal mapping, havingsuccessfully addressed a number of the current challenges associatedwith other particle technologies. This is the first targetednanoparticle that, on the basis of its favorable properties, can be saidto be clinically translatable as a combined optical-PET probe. Thecomplementary nature of this multimodal probe, coupled with its smallsize (˜7-nm diameter), may facilitate clinical assessment by enablingthe seamless integration of imaging data acquired at different spatial,temporal, and sensitivity scales, potentially providing new insightsinto fundamental molecular processes governing tumor biology.

Our in vitro results show receptor-binding specificity of the ˜7-nmtargeted particle probe to M21 and HUVEC cells. Similar findings havebeen reported with receptor-binding assays using the same cell types,but with the monovalent form of the peptide. Cressman, et al., Bindingand uptake of RGD-containing ligands to cellular α_(v)β₃ integrins. IntJ Pept Res Ther. 15, 49-59 (2009). Importantly, the multivalencyenhancement of the cRGDY (SEQ ID NO: 1)-bound particle probe, along withthe extended blood and tumor residence time T_(1/2) values, are keyproperties associated with the particle platform that are not found withthe monovalent form of the peptide.

The relatively long blood T_(1/2) value of 7.3±1.2 hrs estimated for the¹²⁴I-PEG-dot tracer may be related to the chemically neutral PEG-coatedsurface, rendering the probe biologically inert and significantly lesssusceptible to phagocytosis by the reticuloendothelial system. That areduction in the T_(1/2) value to 5.6±0.15 hrs was found for the¹²⁴I-cRGDY-PEG-dot tracer is most likely the result of recognition bytarget integrins and/or more active macrophage activity. However, it issubstantially longer than published blood T_(1/2) values of existingcRGDY peptide (SEQ ID NO: 1) tracers (˜13 minutes), and results ingreater probe bioavailability, facilitating tumor targeting and yieldinghigher tumor uptakes over longer periods of time. Montet, et al.,Multivalent effects of RGD peptides obtained by nanoparticle display. JMed Chem. 49, 6087-6093 (2006). In addition, the tumor T_(1/2) value forthe ¹²⁴I-cRGDY-PEG-dot was about 13 times greater than that for blood,versus only a fivefold difference for the ¹²⁴I-PEG-dot, suggestingsubstantially greater target-tissue localization of the former than thelatter. Such mechanistic interpretations of the in vivo data can beexploited to refine clinical diagnostic, treatment planning, andtreatment monitoring protocols.

The results of this study underscore the clear-cut advantages offered byPET, a powerful, quantitative, and highly sensitive imaging tool fornon-invasively extracting molecular information related to receptorexpression levels, binding affinity, and specificity. The greateraccumulation in and slower clearance from M21 tumors, relative tosurrounding normal structures, allows discrimination of specific tumoruptake mechanisms from non-specific mechanisms (i.e., tissue perfusion,leakage) in normal tissues. A small component of the M21 tumor uptake,however, presumably can be attributed to vascular permeabilityalterations (i.e., enhanced permeability and retention effects).Seymour, Passive tumor targeting of soluble macromolecules and drugconjugates. Crit. Rev. Ther. Drug Carrier Syst. 9, 135-187 (1992). Thisnon-specific mode of uptake reflects a relatively small portion of theoverall tumor uptake at earlier p.i. time points based on the observed %ID/g increases in mice receiving the control tracer (¹²⁴I-PEG-dots, FIG.11B). At 1-hr p.i., no significant % ID/g increases were seen in the M21tumors over the controls. This observation may reflect the effects ofdifferential perfusion in the first hour, with tumor accumulation andretention primarily seen at later p.i. times (i.e., 24 hrs). Further, incomparison with the clinically approved peptide tracer, ¹⁸F-galacto RGD,nearly two-fold greater uptake in M21 tumors was found for the¹²⁴I-cRGDY-PEG-dots34, while additionally offering advantages ofmultivalent binding, extended blood circulation times, and greater renalclearance.

One advantage of a combined optical-PET probe is the ability to assessanatomic structures having sizes at or well below the resolution limitof the PET scanner (i.e., the so-called partial-volume effect), whichmay undermine detection and quantitation of activity in lesions. Forinstance, in small-animal models, assessment of metastatic disease insmall local/regional nodes, important clinically for melanoma stagingand treatment, may not be adequately resolved by PET imaging, given thatthe size of the nodes observed are typically on the order of systemspatial resolution (1-2 mm). By utilizing a second complementary andsensitive imaging modality, near-infrared (NIR) fluorescence imaging,functional maps revealing nodal disease and lymphatic drainage patternscan be obtained. Ballou, et al., Sentinel lymph node imaging usingquantum dots in mouse tumor models. Bioconjugate Chem. 18, 389-396(2007). While further studies investigating the distribution ofintranodal cRGDY-PEG-dot fluorescence in relation to metastatic foci areneeded to determine whether sensitive localization of such foci can beachieved, these results clearly demonstrate the advantages of workingwith such a combined optical-PET probe.

In the clinic, the benefits of such a combined platform for tumorstaging and treatment cannot be overstated. The extended bloodcirculation time and resulting bioavailability of this nanoprobehighlights its use as a versatile tool for both early and long-termmonitoring of the various stages of disease management (diagnosticscreening, pre-treatment evaluation, therapeutic intervention, andpost-treatment monitoring) without restrictions imposed by toxicityconsiderations. An additional important advantage is that while rapidlycleared probes may be useful for certain applications where targettissue localization is itself rapid, localization of many agents inoften poorly vascularized and otherwise relatively inaccessible solidtumors will likely be slow following systemic administration. Thus, thecurrent nanoparticle platform expands the range of applications of suchagents, as the kinetics of target tissue localization are no longerlimiting. Furthermore, deep nodes can be mapped by PET in terms of theirdistribution and number while more precise and detailed localization ofsuperficial nodes can be obtained by NIR fluorescence imaging. Finally,the relatively prolonged residence of the targeted probe from tumorrelative to that from blood, in addition to its multivalencyenhancement, may be exploited for future theranostic applications as aradiotherapeutic or drug delivery vehicle.

EXAMPLE 5 Fluorescent Silica Nanoparticles Conjugated with α_(v)β₃Integrin-Targeting Peptide and/or uMUC1-Targeting Peptide (ThyroidCancer and Squamous Cell Carcinoma (SCC) Models)

A cRGD peptide (Peptides International), having a cysteine endfunctionality, will be attached to the PEG-ylated nanoparticle via athiol-maleimide linkage. The nanoparticles can optionally further befunctionalized by a synthetic peptide ligand, EPPT1. The nanoparticleswill be characterized on the basis of particle size, size distribution,and photobleaching.

Characterization of Nanoparticle-Peptide Conjugates

For assessing photophysical properties on a per-particle basis,spectrophotometry, spectrofluorometry, and multiphoton fluorescencecorrelation spectroscopy (FCS) will be used to determine the particlesize, brightness, and size distribution. Size data will be corroboratedby scanning electron microscopy and dynamic light scattering (DLS)measurements. Ow et al. Bright and stable core-shell fluorescent silicananoparticles. Nano Letters 2005; 5, 113. Average number of RGD peptidesper nanoparticle and coupling efficiency of RGD to functionalized PEGgroups will be assessed colorimetrically under alkaline conditions andBiuret spectrophotometric methods (λ=450 nm, maximum absorbance).

The nanoparticle conjugates will be iodinated via tyrosine linkers tocreate a radiolabeled (¹²⁴I) (T_(1/2)˜4 d) and stable (¹²⁷I) form byusing Iodogen (Pierce, Rockford, Ill.). The end product will be purifiedby using size exclusion chromatography.

Evaluation of In Vitro Targeting Specificity and BiodistributionPatterns of the RGD- and RGD-EPPT-Nanoparticles.

α_(v)β₃ integrin and uMUC1 expression patterns in thyroid and squamouscell carcinoma (SCC) cell lines will be evaluated against known α_(v)β₃integrin-negative and α_(v)β₃ integrin-positive (M21-L and M21 humanmelanoma cell lines, respectively) and uMUC1-negative and uMUC1-positive(U87²⁸,H-29 cell lines, respectively) controls using anti-integrin andanti-uMUC1 antibodies. Cell lines highly expressing α_(v)β₃-integrinand/or MUC1 will be selected for differential binding studies with RGD-and RGD-EPPT-nanoparticles, as well as for in vivo imaging.

Quantitative cell binding assays will assess the labeling efficiency oftumor cells, and biodistribution studies assaying uptake in tumor,organs, and fluids will be performed using radioiodinated nanoparticleconjugates (¹²⁴I-RGD-nanoparticles, ¹²⁴I-RGD-EPPT-nanoparticles). Tocompare PET uptake data of nanoparticle conjugates with that observedinitially using optical NIRF imaging, each nanoparticle conjugate willalso be iodinated to create a radiolabeled (^(124I)) and stable (¹²⁷I)form.

Fluorescence Microscopy with RGD- and RGD-EPPT-C-dots. Differentialbinding of RGD-nanoparticles and RGD-EPPT-nanoparticles to thyroidcarcinoma/SCC cell lines highly expressing α_(v)β₃-integrin and/or MUC1,versus control lines will be visualized by fluorescence microscopy.

Animal models. All animal experiments will be done in accordance withprotocols approved by the Institutional Animal Care and Use Committeeand following NIH guidelines for animal welfare.

In vivo Biodistribution: Male athymic nude mice (6-8 week old, n=5 pertumor) will be subcutaneously (s.c.) injected in both flanks withintegrin-negative/−positive or uMUC1-negative/−positive tumors ofdifferent tissue origins (n=3/each tumor). At 0.5 cm in diameter (i.d.),mice will be injected intravenously (IV) with ¹²⁴I-labeled nanoparticleconjugates (˜500 nm/kg). Animals are sacrificed at 0.5, 1, and 24-hrslater, with removal of tumors, organs, and fluids for weighing andcounting (gamma counter). Biodistribution results will be expressed asthe percentage of injected dose per gram of tissue.

Quantitative Cell Binding Assay. Labeling efficiency will be assessed byincubating fixed numbers of carcinoma cells highly expressingα_(v)β₃-integrin and/or MUC1, with pre-selected concentrations of¹²⁴I-labeled nanoparticle conjugates for 1-hr in a humidified CO₂atmosphere at 37° C. Cells are extensively washed, lysed with 0.1%Triton X, with cell lysates counted in a gamma counter.

Assess of Relative Differences in Tumor-Specific Targeting Using In VivoMultimodality (PET-NIRF) Imaging.

As a high-throughput diagnostic screening tool, optical NIRF imaging canbe used to evaluate relative differences in the biodistribution ofprogressively functionalized nanoparticle conjugates in vivo withincreased sensitivity and temporal resolution. Semi-quantitative data ontumor-specific targeting can also be derived. These preliminary studiesfacilitate the selection of cell lines strongly expressing markers ofinterest for further detailed quantitation of biodistribution andtumor-specific targeting using PET.

Whole-body microPET™ and NIRF optical imaging will be performed over a1-week period to assess differential uptake in flank tumors. The resultsof these studies will be validated with fluorescence microscopy oftumors ex-vivo.

Serial In Vivo NIRF Imaging. Mice will be injected bilaterally withα_(v)β₃ integrin-negative and α_(v)β₃ integrin-positive cells or withuMUC1-negative and uMUC1-positive cells (n=5/tumor). After tumors reach˜0.5 cm i.d., stable iodinated and non-iodinated nanoparticle conjugates(RGD, ¹²⁷I-RGD, RDG-EPPT, ¹²⁷I-RGD-EPPT) will be injected IV. Serialimaging will be performed using the Maestro™ In Vivo FluorescenceImaging System (CRI, Woburn, Mass.) at 0, 0.5, 1, 2, 4, 6, 12, and 24hrs. At 24-h, mice are euthanized, and major tissues/organs dissected,weighed, and placed in 6-well plates for ex-vivo imaging. Fluorescenceemission will be analyzed using regions-of-interest (ROIs) over tumor,selected tissues, and reference injectates, employing spectral unmixingalgorithms to eliminate autofluorescence. Dividing average fluorescenceintensities of tissues by injectate values will permit comparisons to bemade among the various tissues/organs for each injected nanoparticleconjugate.

Dynamic MicroPET Imaging Acquisition and Analysis. Two groups oftumor-bearing mice (n=5/tumor) will be injected with radiolabeled¹²⁴I-nanoparticle conjugates (radiotracers), and dynamic PET imagingperformed for 1-hr using a Focus 120 microPET™ (Concorde Microsystems,TN). One-hour list-mode acquisitions are initiated at the time of IVinjection of ˜25.9 MBq (700 μCi) radiotracers. Resulting list-mode dataare reconstructed in a 128×128×96 matrix by filtered back-projection.ROI analysis of reconstructed images is performed using ASIPro™ software(Concorde Microsystems, TN) to determine the mean and SD of radiotraceruptake (% ID/g) in tumors, other organs/tissues, and left ventricle(LV). Additional data will be obtained from static images at 24-, 48-,and 72-hr post-injection time points. A three-compartment,four-parameter kinetic model will be used to characterize tracerbehavior in vivo. For this analysis, arterial input is measured using anROI placed over the LV.

EXAMPLE 6 Nodal Mapping in Miniswine

Real-time intraoperative scanning of the nodal basin cannot bepractically achieved at the present time, as these systems are generallytoo cumbersome and expensive for use in the operating suite or may beunable to provide the necessary field-of-view or tissue contrast.Further, there are no clinically promising, biostablefluorophore-containing agents, offering improved photophysical featuresand longer circulation lifetimes over parent dyes, available to enhancetissue contrast for extended nodal mapping/resection procedures. Withthis animal study, we will show that advances in both multimodalparticle probes and real-time molecular imaging device technologies canbe readily translated to a variety of future human clinical trials. Suchtransformative technologies can significantly impact standardintraoperative cancer care by providing state-of-the-art targetedvisualization tools for facilitating metastatic SLN detection andenabling accurate delineation of node(s) from adjoining anatomy tominimize risk of injury to crucial structures. Benefits include extendedreal-time in vivo intraoperative mapping of nodal disease spread andtumor extent in the head and neck. Deep nodes can be mapped by PET,while precise and detailed localization of superficial nodes can beobtained by NIR fluorescence imaging. The small size of the particleprobe may also extend the lower limit of nodal sizes that can besensitively detected. The net effect of the proposed non-toxic,multimodal platform, along with the application of combineddiagnostic/treatment procedures, has important implications for diseasestaging, prognosis, and clinical outcome for this highly lethal disease.

Disease Target.

In addition to melanoma, a number of other tumors (i.e., breast, lung,and brain) overexpress αvβ3 integrin receptors and could serve asdisease targets. Metastatic melanoma has a very poor prognosis, with amedian survival of less than 1 year. Successful management relies onearly identification with adequate surgical excision of the cancer.Surgical removal of the primary disease, screening, and treatment forregional lymph node spread is standard-of-care in the US to accuratelystage disease and tailor treatment. The recently revised stagingguidelines recognize the presence of microscopic nodal metastases as ahallmark of advanced stage disease leading to dramatically reducedsurvival. Knowledge of pathologic nodal status is critical for earlyrisk stratification, improved outcome predictions, and selection ofpatient subgroups likely to benefit from adjuvant treatment (therapeuticnodal dissection, chemotherapy) or clinical trials.

Sentinel Lymph Node (SLN) Mapping.

SLN mapping techniques, routinely used in staging melanoma, identify thespecific node(s) that are at highest risk of tumor metastases. Thisprocedure identifies patients harboring metastatic disease for furthertreatment. Standard-of-care techniques rely on injection of radioactivetechnetium (^(99m)Tc) sulfur colloid dye around the primary tumor forSLN localization, followed by the intraoperative use of a gamma probe tomeasure radioactivity in lymphatic structures within an exposed nodalbasin. Blue dye injected about the primary tumor can help delineatesmall SLN(s) from adjacent tissue, but the technique is unreliable andliable to complications. Current SLN mapping and biopsy techniques havelimitations, and account for higher rates of non-localization of SLN(s)in the head and neck compared to other anatomic sites. The head and neckregion is notorious for its unpredictable patterns of metastaticdisease. The close proximity of the primary disease to nodal metastasesin this region makes intraoperative use of the gamma probe difficult dueto interference from the injection site. Importantly, current technologydoes not allow the surgeon to visualize the sentinel node and reliablydifferentiate it from adjoining fat or other tissues, placing vitalstructures (i.e., nerves) at risk for injury during dissection toidentify and harvest this node. The small size of nodes and widevariation in drainage patterns provides additional challenges, resultingin a non-localization rate of around 10%.

Nanoparticles.

The majority of preclinical studies have used RGD peptide orpeptide-conjugate radiotracers as targeting ligands for imagingαvβ3-integrin expression. ¹⁸F-galacto-RGD and ^(99m)Tc-NC100692 arepeptide tracers that have been used successfully in patients to diagnosedisease. Peptide tracers clear rapidly, which may result in reducedreceptor binding and increased background signal from non-specifictissue dispersal. These properties limit the potential of peptidetracers for longer-term monitoring. By contrast, nanoparticle probes(˜10-100 nm), which have also been used for imaging integrin expressionalong tumor neovasculature, have extended circulation half times forperforming longer-term monitoring (i.e., days). Nanoparticles aretypically larger than antibodies and radiopharmaceuticals (<10 kDa), andare associated with slower transmembrane transport, increasedreticuloendothelial system (RES) uptake, and enhanced non-specificuptake due to altered tumor vascular permeability. The 7 nm diametertargeted nanoparticles used for this SLN mapping study are roughlycomparable to the average diameter of an albumin molecule and 2-3 timessmaller than the average diameter of a typical antibody. Relative topeptide tracers, the targeted particle probe is less prone toextravasation and is associated with extended circulation half timesthat enhance tumor targeting. Importantly, 124I-cRGDY-PEG-dotsdemonstrate key in vitro and in vivo properties in M21 tumors necessaryfor clinical translation.

Materials and Methods.

Spontaneous melanoma Sinclair miniature swine (10-12 kg, SinclairResearch Center, MO) were injected intravenously with 5 mCi¹⁸F-fluoro-deoxyglucose (′⁸F-FDG) for whole-body screening of nodaland/or organ metastases. Miniswine underwent 1-hr dynamic ¹⁸F-FDG PETwhole body PET scan using a clinical PET scanner 40 minutes afterinjection to screen for metastatic disease, followed by CT scanacquisition for anatomic localization. Then miniswine were subdermallyinjected in a 4-quadrant pattern about the tumor site (head and necksites preferentially) with multimodal ¹²⁴I-RGD-PEG-dots 48 hrs after¹⁸F-FDG PET, and a second dynamic PET-CT scan performed to assess foradditional nodal metastases.

Miniswine were taken to the operating room for identification of nodes.Optical fluorescence imaging was performed using large field-of-viewnear infrared fluorescence camera system, smaller field-of-view modifiedendoscope, and a modified stereomacroscope for obtaining higherresolution fluorescence images within the exposed surgical bed.

Validation of the fluorescent signal was performed intraoperatively bygamma counting with a clinically-approved hand-held PET device withinthe operative bed to localize targeted dots transdermally, acquiredintraoperatively from skin and the nodes within and nodal basin.

The primary melanoma skin lesion was excised, and an incision made toallow access to the sentinel node(s). Nodal identity was confirmed usinghand held PET and multi-scale optical imaging systems, and the nodes inquestion excised. Specimens were sent for histological assessment formetastases and optical confocal microscopy to confirm the presence ofboth malignancy and nanoparticle fluorescence.

Following harvest of the sentinel nodes, the entire lymph node basin wasexcised and further evaluated using histological methods (withimmunohistochemical markers for melanoma as needed), fluorescencemicroscopy, and the hand-held PET probe for correlative purposes. Thisstep helped identify any other malignant nodes within the nodal basinand the number of ¹²⁴I-RGD-PEG-dots present in adjacent nodes by theirappearance on imaging.

¹²⁴I-RGD-PEG-dots was administered subcutaneously into the limbs of theanimal sequentially. Transit of the ¹²⁴I-RGD-PEG-dots to theinguinal/axillary nodes was followed using the optical imaging systemand hand held PET probes to confirm the duration of transit along thelymphatic pathways. The draining nodal basins was exposed surgically andthe pattern of lymph node drainage observed. The sentinel lymph node washarvested from each site to confirm the lymphatic nature of the tissue.Animals were euthanized, and any further lesions noted on imaging wereexcised in the necropsy room of the animal facility.

Discussion

A whole-body ¹⁸F-fluorodeoxyglucose (¹⁸F-FDG) PET-CT scan revealed aprimary melanomatous lesion adjacent to the spine on the upper back, aswell as a single node in the neck, posteriorly on the right side of theanimal, which were both FDG-avid, and suspicious for metastatic disease.This finding was confirmed after subdermal, 4-quadrant injection of¹²⁴I-RGD-PEG-dots about the tumor site, which additionally identifiedtwo more hypermetabolic nodes, as well as the draining lymphatics. Finalscan interpretation pointed to 3 potential metastatic nodes. Surgicalexcision of the primary lesion, hypermetabolic nodes, and tissue fromother nodal basins in the neck bilaterally was performed after hand-heldPET probes identified and confirmed elevated count rates at the locationof sentinel node(s). Patchy fluorescence signal measured in the excisedright posterior sentinel node tissue correlated with sites of melanomametastases by histologic analysis. All hypermetabolic nodal specimenswere black-pigmented, and found to correlate with the presence ofdistinct clusters of melanoma cells. Thus, the results of surgicallyresected tissue submitted to pathology for H&E and staining for otherknown melanoma markers confirmed multimodal imaging findings.

FIG. 13A shows the experimental setup of using spontaneous miniswinemelanoma model for mapping lymph node basins and regional lymphaticsdraining the site of a known primary melanoma tumor. This intermediatesize miniswine model is needed to simulate the application of sentinellymph node (SLN) biopsy procedures in humans, and more accuratelyrecapitulates human disease. FIG. 13B shows small field-of-view PETimage 5 minutes after subdermal injection of multimodal particles(¹²⁴I-RGD-PEG-dots) about the tumor site. The tumor region, lymph nodes,and the lymphatics draining the tumor site are seen as areas ofincreased activity (black).

FIGS. 14A-14C show whole-body dynamic ¹⁸F-fluorodeoxyglucose (¹⁸F-FDG)PET scan (FIG. 14B) and fused ¹⁸F-FDG PET-CT scans (FIG. 14B)demonstrating sagittal, coronal, and axial images through the site ofnodal disease in the neck. The ¹⁸F-FDG PET scan was performed to mapsites of metastatic disease after intravenous administration and priorto administration of the radiolabeled nanoparticle probe. A singlehypermetabolic node is seen in the neck posteriorly on the right side ofthe animal (arrows, axial images, upper/lower panels), also identifiedon the whole body miniswine image (FIG. 14C).

FIGS. 15A-15C show the same image sets as in FIGS. 14A-14C, but at thelevel of the primary melanoma lesion, adjacent to the spine on the upperback. The PET-avid lesion is identified (arrows, axial images,upper/lower panels), as well as on the whole body miniswine image (FIG.15C).

FIGS. 16A-16C show high resolution dynamic PET (FIG. 16A) and fusedPET-CT images (FIG. 16B) following subdermal, 4-quadrant injection of¹²⁴I-RGD-PEG-dots about the tumor site, simulating clinical protocol,over a 1 hour time period. Three hypermetabolic lymph nodes (arrows)were found in the neck, suggesting metastatic disease. The excised rightposterior SLN was excised and whole body near infrared (NIR)fluorescence imaging was performed. Cy5 fluorescence signal wasdetectable within the resected node (FIG. 16C, top, Cy5 imaging) onwhole-body optical imaging. Pathological analysis of thisblack-pigmented node (arrow, SLN) demonstrated clusters of invadingmelanoma cells on low-(arrows) and high-power cross-sectional views ofthe node by H&E staining (lower two images), and we expect melanomaspecificity to be further confirmed using special stains (Melan A,HMB45, PNL2, and “melanoma associated antigen” biogenex clone NKI/C3).We additionally expect colocalization of the particle with thesemetastatic clusters of cells on confocal fluorescence microscopy andhigh resolution digital autoradiography, confirming metastatic diseasedetection.

EXAMPLE 7 Fluorescent Silica Nanoparticles Conjugated withMC1R-Targeting Peptide (Melanoma Model)

For the multimodality (PET-NIRF) diagnostic imaging experiments, thetargeting peptide and the radiolabel on the nanoparticle surface will beexchanged to determine target specificity, binding affinity/avidity, anddetection sensitivity. Subsequent therapeutic particles will also besynthesized using therapeutic radiolabels (lutetium-177, ¹⁷⁷Lu,t^(1/2)=6.65 d) for targeted killing of MC1R-expressing melanoma cells.Combined quantitative PET and optical imaging findings will becorrelated with tumor tissue autoradiography and optical imaging acrossspatial scales. For cellular microscopy, an in vivo confocalfluorescence scanner for combined reflectance and fluorescence imagingwill be used.

EXAMPLE 8 Fluorescent Nanoparticles for Targeted Radiotherapy

Dose escalation studies with ¹³¹I-RGD nanoparticles will be performedand treatment response will be monitored weekly, over the course of sixweeks, using ^(B)F-FDG PET. Time-dependent tumor uptake and dosimetry ofthe nanoparticle platform will be performed using planar gamma cameraimaging. In vivo imaging data will be correlated with gamma counting ofexcised tumor specimens.

Male nude mice (6-8 wks, Charles River Labs, MA) will be used forgenerating hind leg xenograft models after injection of M21 humanmelanoma cells (5×10⁵ in PBS). Tumors will be allowed to grow 10-14 daysuntil 0.5-0.9 cm³ in size.

¹³¹I-based targeted radiotherapy studies. The therapeutic radionuclide¹³¹I will be used as a radiolabel for targeted radiotherapy. Inestimating the highest possible ¹³¹I dose resulting in no animal deathsand less than 20% weight loss (MTD), a dose escalation study will becarried out in tumor-bearing nude mice. For a 200 rad dose to blood 54,an administered activity of 10 MBq is required, which would deliver adose of 270 rad to tumor. 4 doses of 10 MBq each will be administered toachieve a tumor dose greater than 1000 rad with dose fractionationdesigned to allow repair and sparing of bone marrow. ¹³¹I allows forplanar gamma camera imaging using a pinhole collimator to measure thetime-dependent tumor uptake and dosimetry of the nanoparticles. ¹⁸F-FDGPET allows for quantitative monitoring of tumor response, thus providingcomplementary information.

Based on this data, and in vivo data on the effect of nanoparticlesloaded with paclitaxel, a therapy study with the ¹³¹I-RGD-nanoparticleconjugate will be conducted. Two groups of tumor-bearing mice (n=10 pergroup) will receive either four, 10.4-MBq activities once per week for 4weeks, of i.v.-administered ¹³¹I-RGD-nanoparticle conjugates or salinevehicle (control, n=10), and will be monitored over a 6-week period.Treatment response/progression will be quantified on the basis of tumorvolume (via caliper measurements). All mice from the treatment groupswill also be imaged once per week (˜1 hr sessions) by SPECT imaging(Gamma Medica) over a 6 week period.

¹⁸F-FDG PET Imaging Acquisition and Analysis. Two groups oftumor-bearing mice (n=10/group) will undergo initial PET scanning priorto and then, on a weekly basis after treatment over a 6 week interval.Mice will be injected intravenously (i.v.) with 500 μCi ¹⁸F-FDG andstatic 10-minute PET images will be acquired using a Focus 120 microPET™(Concorde Microsystems, TN) before and after treatment. Acquired datawill be reconstructed in a 128×128×96 matrix by filteredback-projection. Region-of-interest (ROI) analyses of reconstructedimages will be performed using ASIPro™ software (Concorde Microsystems,TN) to determine the mean and SD of radiotracer uptake (% ID/g) intumors. Animals will be sacrificed at the termination of the study andtumors excised for gamma counting.

EXAMPLE 9 Fluorescent Nanoparticles Conjugated with Radionuclide Chelateand MC1R-Targeting Peptide

PEG-ylated nanoparticles will be conjugated with targeting peptides andmacrocyclic chelates binding high-specific activity radiolabels.

High purity two-arm activated commercially available PEGs, derivatizedwith NHS esters or maleimide, will be attached to the silica shell ofthe nanoparticle using standard procedures. Either of the twofunctionalized PEG groups (NHS esters or maleimide) will be availablefor further conjugation with either the peptide-chelate construct,cyclic peptide Re-[Cys3,4,10,D-Phe7]α-MSH3-13 (ReCCMSH(Arg11)), or1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid (DOTA)linker chelators. The covalent attachment of derivatized PEGs to thenanoparticle surface will be performed in such a manner as to exposedifferent functional groups for linking DOTA and peptide-chelateconstructs, as discussed below.

Synthesis and Physicochemical Characterization of FunctionalizedNanoparticles.

Functionalized nanoparticles will be synthesized by establishingcovalent linkages of the following moieties with the derivatized PEGgroups:

(A) DOTA chelates for subsequent high-specific activity radiolabelingwith positron-emitting radiometals (i.e., ⁶⁴Cu) to permit diagnosticdetection with PET imaging. DOTA will be conjugated to thefunctionalized PEGs using standard Fmoc chemistry, and purification ofthe chelated nanoparticles will be performed by chromatography. ⁶⁴Cu and¹⁷⁷Lu will be attached to DOTA by incubation of the reaction mixture at60° C. for 30 min followed by gel filtration or high pressure liquidchromatography purification. Alternatively, PET nuclides, such as ¹²⁴I,⁸⁶Y, ⁶⁸Ga and ⁸⁹Zr, may be conjugated to the nanoparticle, either viathe DOTA-functionalized PEG (radiometals) or tyrosine-functionalized PEG(¹²⁴I). The single photon emitter, ¹⁷⁷Lu, obtained in the form of¹⁷⁷LuCl₃ will be complexed to DOTA for radiotherapy.

(B) αMSH melanoma targeting peptide analogue (ReCCMSH(Arg11)) iscyclized by rhenium. It is necessary to confirm the ratio of DOTAchelates to ReCCMSH(Arg11) moieties on the PEG-ylated nanoparticlesurface.

Characterization of the functionalized nanoparticle preparations will beperformed as follows:

(A) Average number of DOTA chelates per nanoparticle will be determinedby standard isotopic dilution assays with ⁶⁴Cu. Briefly, ⁶⁴Cu will beadded to solutions containing a known amount ofReCCMSH(Arg11)-nanoparticles. Incubated solutions will be spotted onsilica gel-coated glass plates, developed in 1:1 10% ammoniumacetate-to-methanol (with EDTA), and analyzed by radio-TLC. While⁶⁴Cu-labeled ReCCMSH(Arg11)-Nanoparticles will remain at the origin,⁶⁴Cu bound to EDTA will migrate. The percent labeling efficiency will beplotted against total nanomoles of ⁶⁴Cu added to the reaction mixture.The number of chelates attached per nanoparticle can be determined fromthe inflection point of this curve.

(B) Average number of ReCCMSH(Arg11) peptides per nanoparticle andcoupling efficiency of the ReCCMSH(Arg11) to the functionalized PEGgroups will be assessed using spectrophotometric methods (λ=435 nm,maximum absorbance) and the known extinction coefficient ofReCCMSH(Arg11). The incorporation of rhenium offers the advantage thathighly sensitive absorbance measurements of rhenium concentrations canbe made on a small sample of product.

In Vitro and In Vivo Optical-PET Imaging of Multifunctional NanoparticleNanoparticles in Melanoma Models to Assess Tumor-Specific Targeting andTreatment Response.

⁶⁴Cu-DOTA-ReCCMSH(Arg11)-nanoparticles will be compared with the native⁶⁴Cu-DOTA-ReCCMSH(Arg11) construct to test targeting capabilities of thenanoparticles.

Competitive binding assays. The MC1R receptor-positive B16/F1 murinemelanoma lines will be used. The IC₅₀ values of ReCCMSH(Arg11) peptide,the concentration of peptide required to inhibit 50% of radioligandbinding, will be determined using ¹²⁵I-(Tyr2)-NDP7, a radioiodinatedα-MSH analog with picomolar affinity for the MC1R. Single wells will beincubated at 25° C. for 3 h with approximately 50,000 cpm of¹²⁵I-(Tyr2)-NDP in 0.5 ml binding medium with 25 mmol/LN-(2-hydroxyethyl)-piperazine-N-(2-ethanesulfonic acid), 0.2% BSA and0.3 mmol/L 1,10-phenanthroline], with concentrations of (Arg11)CCMSHranging from 10-13 to 10-5 mol/L. Radioactivity in cells and media willbe separately collected and measured, and the data processed to computethe IC₅₀ value of the Re(Arg11)CCMSH peptide with the Kell softwarepackage (Biosoft, MO).

Receptor Quantitation Assay. Aliquots of 5×105 B16/F1 cells will beadded to wells, cultured in 200 μL RPMI media, and incubated at 37° C.for 1.5 h in the presence of increasing concentrations of¹²⁵I-(Tyr2)-NDP (from 2.5 to 100 nCi) in 0.5 mL of binding media (MEMwith 25 mM HEPES, pH 7.4). Cells will be washed with 0.5 mL of ice-cold,pH 7.4, 0.2% BSA/0.01 M PBS twice, and the level of activity associatedwith the cellular fraction measured in a γ-counter. Nonspecific bindingwill be determined by incubating cells and ¹²⁵I-(Tyr2)-NDP withnon-radioactive NDP at a final concentration of 10 μm. Scatchard plotswill be obtained by plotting the ratio of specific binding to free¹²⁵I-(Tyr2)-NDP vs. concentration of specific binding (fmol/millioncells); Bmax, the maximum number of binding sites, is the Xintercept ofthe linear regression line.

B16/F1 murine melanoma lines (5×10⁵ in PBS) will be injectedsubcutaneously into the hind legs of Male nude mice (6-8 week old). Thetumors will be allowed to grow 10-14 days until 0.5-0.9 cm³ in size.

Biodistribution: A small amount of the⁶⁴Cu-DOTA-ReCCMSH(Arg11)-nanoparticle conjugate (˜10 μCi, 0.20 μg) willbe injected intravenously into each of the mice bearing palpable B16/F1tumors. The animals will be sacrificed at selected time points afterinjection (2, 4, 24, 48, 72 hours; n=4-5/time point) and desired tissuesremoved, weighed, and counted for accumulated radioactivity. Additionalmice (n=5) injected with the native radiolabeled construct,⁶⁴Cu-DOTA-ReCCMSH(Arg11) (˜10 μCi, 0.20 μg) will serve as the controlgroup, and evaluated 1 h post-injection. To examine in vivo uptakespecificity, an additional group of mice (2-h time point) will bepre-injected with 20 μg of NDP to act as a receptor block immediatelyprior to the injection of the ⁶⁴Cu-DOTA-ReCCMSH(Arg11) nanoparticleconjugate. Major organs and tissues will be weighed and gamma-counted,and the percentage-injected dose per gram (% ID/g) determined.

Serial In Vivo NIRF Imaging. In parallel with the PET studies below, NIR(fluorescence tomographic imaging, FMT 225, Visen, Woburn, Mass.) willbe performed using a tunable 680 nm scanning NIR laser beam and CCDbefore and after i.v. injection of tumor-bearing animals (n=10). Micewill be kept under continuous isoflurane anesthesia, and placed in aportable multimodal-imaging cassette (compatible with both our FMT 2500and Focus 120 microPET) for FMT scanning before and after injection (1,2, 4, 6, 12, 24, 48 and 72 hours). The NIR fluorescence image, measuredover a 1-10 minute period, will be reconstructed using the Visenproprietary software and superimposed onto a normal photograph of themouse. The imaging data is quantitative, as the measured intensity isdirectly related to the NIR fluorophore concentration, enablingparametric maps of absolute fluorophore concentrations to be generatedfor co-registration with the acquired PET imaging data.

Dynamic PET Imaging Acquisition and Analysis. Two groups oftumor-bearing mice (n=5/group) will be placed in the imaging cassettefor co-registering sequential PET-optical studies. Mice will be injectedintravenously (i.v.); one with radiolabeled ⁶⁴Cu-DOTA-ReCCMSH(Arg11)nanoparticle conjugates and the second with native⁶⁴Cu-DOTA-ReCCMSH(Arg11) constructs. Following injection, dynamic 1-hrPET images will be acquired using a Focus 120 microPET™ (ConcordeMicrosystems, TN). One-hour list-mode acquisitions are initiated at thetime of IV injection of radiolabeled probe (˜1 mCi). Resulting list-modedata will be reconstructed in a 128×128×96 matrix by filteredback-projection. Region-of-interest (ROI) analyses of reconstructedimages are performed using ASIPro™ software (Concorde Microsystems, TN)to determine the mean and SD of radiotracer uptake (% ID/g) in tumors,other organs/tissues, and left ventricle (LV). Tracer kinetic modelingof the data will permit estimation of pharmacokinetic parameters,including delivery, clearance, and volume of distribution. As noted, anarterial blood input is measured using an ROI placed over the LV (as ameasure of blood activity). Additional data will be obtained from staticimages at 24 hr, 48 hr, 72 hr post-injection time points.

Fluorescence microscopy and autoradiography of tissues. A combination ofoptical imaging technologies exhibiting progressively smaller spatialscales (i.e., whole body fluorescence imaging, fluorescence macroscopy,and in vivo fluorescence confocal laser scanning microscopy) will beutilized for imaging tumors in live, intact animals at 72-hpost-injection. Mice will be maintained under continuous isofluoraneanesthesia, thus enabling detection and localization of fluorescencesignal from the whole animal/organ level to the cellular level over arange of magnifications. Whole animal/macroscopic imaging will beperformed with fluorescence stereomicroscope (Visen; Nikon SMZ1500)fitted with Cy5 fluorescence filter sets and CCD cameras. Fluorescenceconfocal laser scanning microscopy capabilities will be developed. Micewill subsequently be euthanized for autoradiography in order to maptracer biodistributions at high resolution throughout the tumor volume.Tumors will be excised, flashfrozen, serially sectioned (1□0μ sections)and slide-mounted, with alternating slices placed in contact with aphosphor plate in a light-tight cassette (up to 1 wk). H&E staining willbe performed on remaining consecutive sections. Autoradiographicfindings will be correlated with PET imaging data and histologicalresults.

The therapeutic radionuclides ¹⁷⁷Lu or ⁹⁰Y may alternatively be used fortargeted radiotherapy. In estimating the highest possible ¹⁷⁷Lu doseresulting in no animal deaths and less than 20% weight loss (MTD), adose escalation study will be carried out in tumor-bearing nude mice.Doses of radiopharmaceutical suspected to be at (or near) the MTD basedon literature values for ¹⁷⁷Lu will be evaluated.

EXAMPLE 10 Fluorescent Nanoparticles Functionalized to Conjugate withLigand and Contrast Agent Via “Click Chemistry”

Synthesis of Nanoparticles Containing Versatile Functional Groups forSubsequent Conjugation of Ligand (e.g., Peptides) and Contrast Agent(e.g., Radionuclides).

In order to synthesize an array of nanoparticle-peptide-chelateconstructs suitable for high-specific activity radiolabeling, a“click-chemistry” approach may be used to functionalize the nanoparticlesurface (FIG. 17). This method is based on the copper catalyzedcycloaddition of azide to a triple bond. Such an approach would allowfor a great deal of versatility to explore multimodality applications.

Nanoparticle synthesis and characterization. The PEG groups that will becovalently attached will be produced following the scheme in FIGS.14A-14C. PEG will be covalently attached to the nanoparticle via thesilane group. Standard chemical pathways will be used for the productionof the functionalized PEG with triple bonds.

Functionalization of nanoparticles with triple bonds. To synthesize thebifunctionalized PEGs, the first step will employ the well studiedreaction of activated carboxylic ester with aliphatic amine (FIG. 18).Alternatively, another suitable triple-bond bearing amine, for example,p-aminophenylacetylene, can be used. The second step of the synthesisalso relies on a well-known conjugation reaction.

Synthesis and Physicochemical Characterization of FunctionalizedNanoparticles Conjugated with Model Peptides and Chelates.

The functionalized nanoparticle contains both (A) desferrioxamine B(DFO) for subsequent high-specific activity radiolabeling with thepositron-emitter zirconium-89 (⁸⁹Zr) and (B) the SSTR-targeting peptide,octreotate.

Synthesis of DFO with an azide bond. DFO with an azide group will beproduced by reaction of DFO-B with pazido benzoic acid) (FIG. 19) andpurified. The “click chemistry” reaction is a 1,3-dipolar cycloadditionat room temperature and the conditions are often referred to as “HuigsenConditions”. Although the reactions can generally be completed at roomtemperature in ethanol, it may be appropriate to heat the reaction. Thecatalyst is often Cu(I)Br, but alternatives include Cu(I)I or Cu(II)SO4(with a reductant). Knor et al. Synthesis of novel1,4,7,10-tetraazacyclodecane-1,4,7,10-tetraacetic acid (DOTA)derivatives for chemoselective attachment to unprotectedpolyfunctionalized compounds. Chemistry, 2007; 13:6082-90. Clickreactions may also be run in the absence of any catalyst. Alternatively,the NH³⁺ group in DFO-B may be converted directly into an azide group.

Synthesis of Tyr3-octreotate with an azide. Solid phase peptidesynthesis (SPPS) of Tyr3-octreotate (FIG. 20A) will be performed on apeptide synthesizer. Briefly, the synthesis will involve the Fmoc(9-fluorenylmethoxycarbonyl) method as previous described for thispeptide. Briefly, the instrument protocol requires 25 μmol of subsequentFmoc-protected amino acids activated by a combination of1-hydroxybenzotriazole (HOBt) and2-(1Hbenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate(HBTU). The Fmoc-protected amino acids will be purchased commerciallyunless otherwise stated; the pre-packed amino acids will be obtainedfrom Perkin-Elmer (Norwalk, Conn.), while those unavailable inpre-packed form, such as the Damino acids and Fmoc-Cys(Acm) will besupplied by BACHEM Bioscience, Inc. (King of Prussia, Pa.) orNovabiochem (San Diego, Calif.). The azide group (for the “click”chemistry) will be introduced into the peptide backbone via coupling ofan azide-containing acid to the N-terminus of the peptide, while thepeptide is still protected and attached to the resin (FIG. 20B).

Synthesis of functionalized nanoparticles. The next step will be toconjugate both the DFO having an azide bond and Tyr3-octreotate havingan azide bond (FIGS. 21A and 21B) to the nanoparticle. “Click chemistry”is highly selective, quantitative and can be performed very fast andusing mild conditions. The number of combined azide groups from DFO andTyr3-octreotate will be controlled to never exceed the number ofavailable triple-bonds; the triple bonds will always be in <5% excess.

Functionalized nanoparticle characterization. Average number of DFOchelates peptide per nanoparticle will be determined by performing astandard isotopic dilution assay with ⁸⁹Zr (or ⁶⁸Ga). ⁸⁹Zr will beproduced on cyclotron and purified. Briefly, 10 concentrations of89Zr-oxalate will be added to solutions containing a known amount ofDFO-derived nanoparticles. Following a 30 min. room temperatureincubation, the solutions will be spotted on silica gel coated glassplates, developed in 1:1 10% ammonium acetate-to-methanol (with EDTA)and analyzed by radio-TLC. Whereas the ⁸⁹Zr-DFO-derived nanoparticleswill remain at the origin, nonspecifically bound ⁸⁹Zr bound to EDTA willmigrate. The percent labeling efficiency will be plotted as a functionof total nanomoles of ⁸⁹Zr added to the reaction mixture. The number ofchelates attached to the nanoparticle can then be determined from theinflection point of this curve.

Average number of Tyr3-octreotate peptide per nanoparticle will bedetermined by assaying the disulfide bridge of Tyr3-octreoate. Briefly,the disulfide bonds of the Tyr3-octreotate can be cleaved quantitativelyby excess sodium sulfite at pH 9.5 and room temperature. DTNB or Elman'sreagent can be used to quantitate thiols in proteins by absorptionmeasurements. It readily forms a mixed disulfide with thiols, liberatingthe chromophore 5-merapto-2-nitrobenzoic acid (absorption maximum 410nm). Only protein thiols that are accessible to this water-solublereagent are modified. Alternatively, the Measure-iT™ Thiol Assay Kitfrom Invitrogen can be used.

In Vivo Testing in Suitable Tumor Models.

Subcutaneous xenograft models using AR42J tumor-bearing female SCID micewill be generated. Briefly, AR42J cells (1×10⁷), will be injectedsubcutaneously into the flanks of female SCID mice. The tumors will beallowed to grow 10-12 days until 0.5-0.9 cm³ in size.

Radiolabeling of the DFO-nanoparticle by ⁸⁹Zr is expected to proceed in<15 min. at room temperature. Non-specifically bound ⁸⁹Zr will beremoved by addition of EDTA followed by a gel filtration step.

Receptor binding assays. The receptor binding assays will be performedusing ⁸⁹Zr-DFO-nanoparticles on membranes obtained from AR42J tumors.The competing ligands, natZr-DFO-Nanoparticles and natZr-DFO-octreotatewill be prepared by the reaction of high purity natural zirconiumoxalate with DFO-octreotate and DFO-Nanoparticles, respectively. Purityof the final products will be confirmed by HPLC. IC50 values will bedetermined according to previously published methods, using theMillipore MultiScreen assay system (Bedford, Mass.). Data analysis willbe performed using the programs GraFit (Erithacus Software, U.K.),LIGAND (NIH, Bethesda, Md.), and GraphPad PRISM™ (San Diego, Calif.).

In vitro assays. The AR42J cells will be harvested from monolayers withCell Dissociation Solution (Sigma Chemical Co., St. Louis, Mo.) andresuspended in fresh DMEM media at a concentration of 2×106 cells/mL. Analiquot of about 0.3 μmol of ⁸⁹Zr-DFO-nanoparticles will be added to 10mL of cells, incubated at 37° C. with continuous agitation. At 1, 5, 15,30, 45, 60 and 120 min triplicate 200-μL aliquots will be removed andplaced in ice. The cells will immediately be isolated by centrifugation,and the % uptake of the compound into the cells will be calculated.

Biodistribution. A small amount of the ⁸⁹Zr-DFO-nanoparticles (˜10 μCi,0.20 μg) will be injected intravenously into each of the mice bearingpalpable AR42J-positive tumors. The animals will be sacrificed atselected time points after injection (1, 4, 24, 48, 72 hours; n=4-5) anddesired tissues will be removed, weighed, and counted for radioactivityaccumulation. Two additional control groups will be studied at 1 hpost-injections: (A) mice injected with the native radiolabeled peptide⁸⁹Zr-DFO-octreotate (˜10 μCi, 0.20 μg), and (B) mice pre-injected with ablockade of Tyr3-octreotate (150 μg) to demonstrate receptor-mediatedaccumulation of the ⁸⁹Zr-DFO-nanoparticles. Tissues including blood,lung, liver, spleen, kidney, adrenals (STTR positive) muscle, skin, fat,heart, brain, bone, pancreas (STTR positive), small intestine, largeintestine, and AR42J tumor will be counted. The percentage injected doseper gram (% ID/g) and percentage injected dose per organ (% ID/organ)will be calculated by comparison to a weighed, counted standardsolution.

In vivo NIRF imaging. Serial imaging will be performed using theMaestro™ In Vivo Fluorescence Imaging System (CRI, Woburn, Mass.) at 0,0.5, 1, 2, 4, 6, 12, 24, 48 and 72 hrs. At 72-hr, mice will beeuthanized, and major tissues/organs dissected, weighed, and placed in6-well plates for ex-vivo imaging. Fluorescence emission will beanalyzed using regions-of-interest (ROIs) over tumor, selected tissues,and reference injectates, employing spectral unmixing algorithms toeliminate autofluorescence. Fluorescence intensities and standarddeviations (SD) will be averaged for groups of 5 animals. Dividingaverage fluorescence intensities of tissues by injectate values willpermit comparisons to be made among the various tissues/organs for eachinjected nanoparticle conjugate.

In vivo small animal PET imaging. Small animal PET imaging will beperformed on a microPET®-FOCUS™ system (Concorde Microsystems Inc,Knoxville Tenn.). Mice bearing the AR42J tumors (n=5 per group) will beanesthetized with 1-2% isoflurane, placed in a supine position, andimmobilized in a custom prepared cradle. The mice will receive 200 μCiof the ⁸⁹Zr-DFO-octreotate-nanoparticle complex via the tail vein andwill be imaged side by side. Animals will initially be imaged byacquiring multiple, successive 10-minute scans continuously from thetime of injection over a 1-hr time frame, followed by 10-min static dataacquisitions at 2, 4, 24, 48 and 72-hrs post-injection. Standard uptakevalues (SUVs) will be generated from regions of interest (ROIs) drawnover the tumor and other organs of interest. Co-registration of the PETimages will be achieved in combination with a microCAT-II camera (ImtekInc., Knoxville, Tenn.), which provides high-resolution X-ray CTanatomical images. The image registration between microCT and PET imageswill be accomplished by using a landmark registration technique andAMIRA image display software (AMIRA, TGS Inc, San Diego, Calif.). Theregistration method proceeds by rigid transformation of the microCTimages from landmarks provided by fiducials directly attached to theanimal bed.

Pharmacokinetic measurements. The biodistribution and dynamic PET datawill provide the temporal concentration of⁸⁹Zr-DFO-octreotate-nanoparticle in tissue which will allow forcharacterization of pharmacokinetic parameters of the agent.

Fluorescence microscopy and autoradiography of tissues ex vivo.Localization of nanoparticle conjugates in tissues will be performed onfrozen sections. Imaging by microPET will allow us to evaluate fully theglobal distribution in tumors and other non-target tissues. Followingthe acute stage of the imaging trial, autoradiography will also beperformed on the tumors, and this data will be correlated to both thePET imaging and histological results. Consecutive slices (˜10 μm) willbe taken, alternating slices for autoradiography and for histologicalanalysis. These sections will also be analyzed by multichannelfluorescence microscopy in the NIR channel.

EXAMPLE 11 Particle Internalization Studies

The goal of this study is to evaluate the binding and internalization ofthe present nanoparticles to assess their localization in subcellularorganelles and exocytosis. This will help study the fate offunctionalized particles with different targeting moieties and attachedtherapies. For example, both diagnostic nanoparticles (e.g.,non-targeted PEG-coated versus cRGD-PEG-coated nanoparticles) andtherapeutic nanoparticles (e.g., cRGD-PEG-nanoparticles attached toiodine for radiotherapy, attached to tyrosine kinase inhibitors, orattached to chemotherapeutic drugs such as Taxol.)

Materials and Methods

Internalization/uptake studies. Internalization assays andcolocalization studies were performed for identifying specific uptakepathways.

Melanoma cells, including human M21 and mouse B16 cells (˜2×10⁵cells/well), were plated in 8-well chamber slides (1.7 cm²/well) slidesor 24 well plates (1.9 cm²/well) with a 12 mm rounded coverglass andincubated at 37° C. overnight. To monitor targeted nanoparticleinternalization, cells were incubated with cRGD-PEG dots (0.075 mg/ml)for 3 hrs at 37° C. To remove unbound particles in the medium, cellswere rinsed twice with PBS. Confocal microscopy was performed on a Leicainverted confocal microscope (Leica TCS SP2 AOBS) equipped with a HCX PLAPO: 63×1.2 NA Water DICD objective to assess co-localization ofcRGD-PEG-dots with organelle-specific stains or antibodies. Images wereanalyzed using ImageJ software version 1.37 (NIH Image;http://rsbweb.nih.gov/ij/).

Co-localization Assays/Dye-bound markers. In order to identify endocyticvesicles involved in C dot internalization, colocalization assays inliving cells were performed using dye-bound markers. Cells werecoincubated with nanoparticles and different dyes. The dyes include: 100nM Lysotracker red for 30 min to label acidic organelles along endosomalpathway; 2 μg/mL transferrin Alexa 488 conjugate to label recycling andsorting endosomes (clathrin-dependent pathway); 1 mg/mL 70 kDadextran-FITC conjugate at 37° C. for 30 min to label macropinosomes.

Co-localization/Organelle-specific antibodies. Immunocytochemistry willbe performed with known markers for Golgi and lysosomes. For Golgi,Giantin (Abcam, rabbit polyclonal, 1:2000) will be used for human cells;GM-130 (BD Pharmingen, 1 μg/ml) will be used for mouse cells. ForLysosomes, LC3B (Cell Signaling, rabbit polyclonal, 0.5 μg/ml) will beused.

For Giantin or LC3B staining, cells will be blocked for 30 minutes in10% normal goat serum/0.2% BSA in PBS. Primary antibody incubation(rabbit polyclonal anti-Giantin antibody (Abcam catalog # ab24586,1:2000 dilution) or LC3B (Cell Signaling, C#2775, 0.5 ug/ml) will bedone for 3 hours, followed by 60 minutes incubation with biotinylatedgoat anti-rabbit IgG (Vector labs, cat #:PK6101) in 1:200 dilution.Detection will be performed with Secondary Antibody Blocker, Blocker D,Streptavidin-HRP D (Ventana Medical Systems) and DAB Detection Kit(Ventana Medical Systems) according to manufacturer instructions.

For GM-130 staining, cells will be blocked for 30 min in Mouse IgGBlocking reagent (Vector Labs, Cat#: MKB-2213) in PBS. The primaryantibody incubation (monoclonal anti-GM130, from BD Pharmingen;Cat#610822, concentration 1 ug/mL) will be done for 3 hours, followed by60 minutes incubation of biotinylated mouse secondary antibody (VectorLabs, MOM Kit BMK-2202), in 1:200 dilution. Detection will be performedwith Secondary Antibody Blocker, Blocker D, Streptavidin-HRP D (VentanaMedical Systems) and DAB Detection Kit (Ventana Medical Systems)according to manufacturer instructions.

For temperature-dependent studies, nanoparticles will be incubated withcRGD-PEG-nanoparticles at 4° C., 25° C., and 37° C. to assess fractionof surface bound versus internalized particles.

For exocytosis studies, nanoparticles (0.075 mg/ml) will be incubatedfor 4 hours and chamber slides washed with PBS, followed by addition offresh media. At time intervals of 0.5, 1.0, 1.5, 2.5, 4.5, 8.0 hrs,cells will be washed, typsinized, and fluorescence signal of cells andmedia measured by fluorimetry. In dose-response studies, cells will beincubated over a range of concentrations and incubation times, andassayed using flow cytometry. In viability studies, cell viability willbe measured using a trypan blue exclusion assay before and afterincubation to assess for toxicity. In time-lapse studies, mechanism ofnanoparticle internalization in living cells will be investigated afterincubating cells with nanoparticle conjugates at different temperaturesof incubation (4° C., 25° C., and 37° C.) using an inverted confocalmicroscope over a 12-hr period at 20 min intervals.

Discussion

cRGD-PEG-dots and PEG-dots were found to co-localize with LysotrackerRed in M21 and B16 cells suggesting uptake in the endosomal pathway(FIG. 22). Data showed that these particles strongly colocalize withtransferrin and dextran. Regardless of surface functionality and totalcharge, nanoparticles (6-7 nm in hydrodynamic diameter) studied appearedto follow the same route. Time lapse imaging in both cell typesdemonstrated internalization of functionalized nanoparticles within asmall fraction of the plated cells. Particles were eventually deliveredto vesicular structures in the perinuclear region. Colocalization assayswith Giantin (or GM-130) is not expected to show nanoparticlefluorescent signal in the Golgi.

EXAMPLE 12 Dual-Modality Silica Nanoparticles for Image-GuidedIntraoperative SLN Mapping and Interventions

Dual-Modality Silica Nanoparticles for Image-Guided Intraoperative SLNMapping

These studies were expanded to include optical imaging using theportable ArteMIS™ fluorescence camera system, along with radiodetectionusing the gamma probe, for performing real-time assessments of thedraining tumor lymphatics and nodal metastases, as well as assessment oftumor burden. In a representative miniswine (FIGS. 23A-23I), initialpreoperative PET-CT scanning was performed using ¹⁸F-FDG and¹²⁴I-cRGDY-PEG-C dots using the foregoing imaging procedure. Axial CTimages revealed a primary pelvic tumor (FIG. 23A) and draining SLN (FIG.23B), which were seen as areas of increased activity on thecorresponding ¹⁸F-FDG PET scan (FIG. 23C, FIG. 23D). These findings wereconfirmed 2 days later by dynamic PET-CT imaging about 5 minutes aftersubdermal, 4-quadrant injection of the particle tracer about the tumorsite; coregistered axial (FIGS. 23E, 23G) and coronal (arrows, FIGS.23F, 23H) views demonstrate these findings. Following pre-operativescanning, the skin overlying the SLN site was marked for intraoperativelocalization, and the miniswine was transported to the intraoperativesuite. Baseline activity measurements, made over the primary tumor andSLN sites using the portable gamma probe (FIG. 23I), showed a 20-foldincrease in activity within the SLN relative to background signal.

For real-time optical imaging of the lymphatic system, a secondsubdermal injection of ¹²⁴I-cRGDY-PEG-C dots was administered about thetumor site with the skin intact, and the signal viewed in the color(FIG. 24A) and Cy5.5 fluorescent channels (FIG. 24B). The adjacent nodalbasin was exposed, and fluorescent signal was seen in the NIR channelflowing from the injection site (FIG. 24C) into the main proximal (FIGS.24C, 24D), mid (FIG. 24E), and distal (FIG. 24F) lymphatic branches,which drained towards the SLN (FIG. 24F). Smaller caliber lymphaticchannels were also visualized (FIGS. 24D, 24E). The black-pigmented SLN,viewed in dual-channel mode (FIGS. 24G, 24H), was further exposed (FIG.24I) prior to successive nodal excision (FIGS. 24J-24M). Fluorescencesignal within the in situ (FIG. 24K) and ex vivo (FIG. 24M) nodalspecimen was confirmed by gamma emissions using the gamma probe (FIG.24I), and seen to correspond to scattered clusters of tumor cells onlow-power (box, FIG. 24N) and high-power (FIG. 24O) views fromH&E-stained tissue sections. Positive expression of HMB45 was identifiedon low-power (FIG. 24P) and high-power (FIG. 24Q) views, consistent withmetastatic melanoma.

Surprisingly, and by contrast to the observed ¹⁸F-FDG findings,¹²⁴I-RGD-PEG-C dots were found to specifically discriminate betweenmetastatic tumor infiltration and inflammatory processes in theseminiswine. Mechanistic differences in the behavior of these agents atthe cellular and subcellular levels, as well as the presence of anintegrin-targeting moiety on the particle surface, may account for theobserved imaging findings. In multiple miniswine harboringpathologically-proven inflammatory changes due to granulomatous disease(n=3), ¹⁸F-FDG failed to detect metastatic disease, while identifyinginflammatory and other metabolically active sites. These discrepantfindings highlighted the ability of the particle tracer to selectivelytarget, localize, and stage metastatic disease, while ¹⁸F-FDG failed inmany cases to accurately stage cancer spread, instead identifying sitesof inflammation.

In a representative miniswine study illustrating these findings, initialaxial ¹⁸F-FDG PET-CT scans showed calcification within the leftposterior neck on CT (FIG. 25A), corresponding to an area of intenseactivity on the ¹⁸F-FDG PET (FIG. 25B). Low-power (FIG. 25C) andhigh-power (FIG. 25D) views of H&E stained tissue sections revealeddiffuse inflammatory changes, consistent with granulomatous disease.Intense ¹⁸F-FDG PET activity was additionally seen within themetabolically active bone marrow compartment of these young miniswine(FIGS. 25A, 25B). By contrast, the particle tracer imaging studyidentified bilateral metastatic neck nodes. A right neck node on axialCT imaging (FIG. 25E) was seen to be PET-avid on co-registered PET-CT(FIG. 25F); additional bilateral nodes on a more superior CT image (FIG.25G) were also hypermetabolic on fused PET-CT (FIG. 25H). Moreover, leftneck calcifications (FIGS. 25E, 25G) showed no PET activity onco-registered scans (FIGS. 25F, 25H). Corresponding H&E-stained SLNtissue sections revealed dark melanomatous clusters on low-power (box,FIG. 25I) and high-power views (FIG. 25J), seen to be comprised ofmelanoma cells and melanophages. A single frame (FIG. 25K) selected from3D PET reconstructed images again illustrated multiple, bilateralPET-avid neck nodes and associated draining lymphatic channels.Importantly, bulk activity was seen in the bladder 1 hr post-injectionwithout significant tracer accumulation over the liver region.

The above findings were seen to better advantage on PET-CT fusion MIPimages generated from dynamic imaging data sets acquired over a 1 hourperiod after ¹⁸F-FDG (FIG. 26A) or local particle tracer administration(FIGS. 26B, 26C). For ¹⁸F-FDG, a clear absence of nodal metastases isnoted, with diffusely increased activity seen withinmetabolically-active bony structures. In contrast to these findings,¹²⁴I-cRGDY-PEG-C dots detected bilateral metastatic neck nodes, alongwith draining lymphatic channels.

Dual-Modality Silica Nanoparticles for Image-Guided Interventions:Treatment Response.

The ability of the particle tracer to discriminate metastatic diseasefrom tissue inflammatory changes could potentially be exploited in avariety of therapeutic settings—either surgically-based orinterventionally-driven—as treatment response assessments are oftenconfounded by the presence of inflammatory changes, makinginterpretation difficult. Image-guided interventions, such astherapeutic tumor ablations, may specifically benefit from theinnovative coupling of new particle platform and imaging devicetechnologies to (1) enable earlier post-procedural evaluation ofresponse; (2) verify complete ablation or detect residual tumorrepresenting treatment failure, and (3) improve tumor surveillancestrategies. Locally ablative therapies, including microwave ablation,cryoablation, radiofrequency ablation

(RFA), and laser interstitial therapy, induce local thermal injury viaan energy applicator insertion into tumors. These methods are typicallyemployed as alternative options in patients deemed ineligible forsurgical excision. Further, patients undergoing ablative therapies areoften poor surgical candidates due to co-morbidities. Widely used inclinical practice, they offer a distinct advantage, as they can beperformed percutaneously as outpatient procedures with significantlyless morbidity, and may improve quality of life and survival in selectedpatient cohorts.

Accurate post-therapy imaging, typically acquired 1-3 months after anablation procedure, traditionally utilized contrast enhanced volumetricimaging, such as CT or Mill. These techniques suffer from a number ofdrawbacks. First, they are limited to identifying the presence ofabnormal enhancement or growth in the size of the tumor area, consideredprimary indicators of residual tumor or recurrent disease. Diffuse rimenhancement about the ablation zone on post-procedural evaluations maybe related to inflammation and hyperemia in the ablation zone, and oftendoes not necessarily represent residual tumor. Increasing enhancement,notably irregular or nodular, is considered suspicious for tumor.However, these interpretations are controversial, as an ablation zonecan look larger than expected for several months post-procedure, andenhancement might also reflect granulation or scar tissue formation.

Functional methods, such as ¹⁸F-FDG PET, have also been used to assessthe efficacy and effects of locally ablative procedures, but may sufferfrom an inability to accurately discriminate tumor from inflammatorychanges. Thus, interpretation of imaging changes (i.e., inflammation,tumor) at the tissue level in response to ablative procedures usingcurrent morphologic or functional assessments, particularly at earlytime intervals, is a significant challenge. What is needed are reliableendpoints for ablation success and unequivocal detection of residualdisease in the postablation period.

As a forerunner to performing future ablations of metastatic liverlesions, a proof-of-concept radiofrequency ablation (RFA) study of alarger (i.e., 1-2 cm) SLN was performed in a miniswine with metastaticmelanoma to evaluate early treatment response in the presence of theparticle tracer. PET-CT imaging findings prior to and after RFA werecorrelated histologically. Following subdermal injection of¹²⁴I-cRGDY-PEG-C dots (˜0.6 mCi) about the primary left pelvic tumor, aninitial baseline coronal CT showed a 2.2×1.6 cm SLN (FIG. 27A) superiorto the tumor site, which was PET-avid (FIGS. 27B, 27C). Thehypermetabolic left pelvic tumor is also shown (FIG. 27B), notingadditional particle tracer flow within a draining lymphatic channel onfused PET-CT images (FIGS. 27C, 27D). Additional serial CT scans wereacquired to localize the node (FIG. 27E) prior to the ablation procedureand guide RFA probe insertion (FIG. 27F) into the node (below level ofcrosshairs). On the corresponding pre-ablation co-registered PET-CTscan, the PET-avid SLN was seen just posterior to crosshairs (FIG. 27G).A partial node ablation was performed for 12 minutes using a 2 cm activetip RFA probe (Cool-tip ablation system, Covidien plc, Dublin, Ireland).Post-ablation PET-CT showed mildly reduced tracer activity in theablation zone, anterior to the electrode tip (FIG. 27H). Pre- andpost-ablation imaging findings were confirmed histologically. H&Estaining of pre-ablated core biopsy tissue from the SLN confirmeddiffuse metastatic tumor infiltration on low-power (FIG. 27I) andhigh-power (FIG. 27J) views. Post ablation, the extent of metastaticinfiltration decreased on H&E stained nodal tissue, seen oncorresponding low—(FIG. 27K) and high-power views (FIG. 27L).Coagulative necrosis and lymphoid tissue were also identified, alongwith multifocal hemorrhages (FIGS. 27K, 27L, respectively). TUNELstained high-power views prior to ablation reveal scattered neoplasticcells (FIG. 27M). On post-ablation TUNEL staining, focal areas ofnecrosis (red) were seen on both low—(FIG. 27N) and high-power (FIG.27O) views.

CONCLUSIONS

Lymph node metastases are a powerful predictor of outcome for melanoma.Early detection of micrometastases in regional lymph nodes using SLNmapping may permit the timely stratification of patients to appropriatetreatment arms, and can potentially improve patient outcomes. Althoughthe current standard-of-care SLN mapping and biopsy techniques rely onthe use of radioactivity-based identification of SLNs, a number oflimitations of this technology exist. These include low spatialresolution, reduced staging accuracy, absence of target specificity,slow tracer clearance that may obscure the surgical field, and the lackof accurate intraoperative visualization to prevent injury to vitalstructures lying in close proximity to SLNs.

The recent introduction of newer generation, biocompatible particleplatforms that can be actively tailored and refined to overcome thesedrawbacks according to key design criteria, while enabling selectiveprobing of critical cancer targets, can offer important insights intocellular and molecular processes governing metastatic disease spread.The additional adaptation of such platforms for multimodality imagingcould be used to advantage by the operating surgeon or interventionalistto explore these processes in a variety of image-guided proceduralsettings.

One such dual-modality platform, a clinically-translatedintegrin-targeting silica nanoparticle developed for both optical andPET imaging, meets a number of key design criteria—small size, superiorbrightness, enhanced tumor tissue retention, and low backgroundsignal—that make it an ideal agent for SLN localization and stagingduring SLN biopsy procedures when coupled with portable, real-timeoptical camera systems. The ability to discriminate metastatic diseasefrom tissue inflammatory changes in melanoma models, which are oftenco-existing processes, may provide a more accurate and reliable markerfor the assessment of treatment response in the future. Furtherinvestigation in a broader set of cancer types and treatments iswarranted using either surgically-based or interventionally-driventherapies.

EXAMPLE 13 SLN Mapping of Prostate Cancer

Multimodal nanoparticle bearing HuJ591-F(ab′)2 fragments are noveldiagnostic probes for binding prostate specific membrane antigen (PSMA).By synthesizing particles bearing multiple F(ab′)2 fragments, we can (1)enhance binding affinity/potency due to multivalency effects and (2)alter in vivo distributions, as clearance and uptake will be dominatedby particle kinetic behavior, rather than the antibody itself. Bymaintaining particle size below or just at the renal cut-off of 10-nmdiameter, renal clearance is promoted. Further, target-to-backgroundratios will increase on the basis of improvements in (1) and (2),potentially improving diagnostic specificity and disease staging.

Synthesis/Characterization of ¹²⁴I-J591 F(Ab′)2-Bound Particles.

To generate PEGylated F(ab′)2 constructs, 100 ug of F(ab′)2 was added toan eppendorf tube in 100 μl PBS, followed by incubation with 4 μl of 2mg/ml Traut's reagent for 1 h at room temperature. Maleimide-PEG (6 mg)dissolved in buffer solution was then added. The solution was incubatedovernight and purified with a PD-10 column prior to particle attachment.F(ab′)2 fragments are being radiolabeled with iodine-124 (¹²⁴I) tocreate a dual-modality particle platform, and the specific activity,purity, and radiochemical yield will be derived. Particle size andconcentration will additionally be assessed by FCS.

EXAMPLE 14 in-Human Dual-Modality Silica Nanoparticles forIntegrin-Targeting in Melanoma

Nanomaterials, in particular nanoparticle probes, possess uniquephysicochemical and biological properties, as well as surfaceversatility. By exploiting their surface versatility, biocompatible,multifunctional particles can be selectively modified with molecularmarkers that recognize and localize key cancer targets. Of increasingimportance in operative settings is the need for more robustoptically-driven multifunctional particle probes which can improvereal-time targeted detection of local/regional disease spread about theprimary tumor site, thus facilitating treatment. Real-time delineationof disease from critical neural and/or vascular structures inintraoperative settings will also be paramount. Duncan, R, The dawningera of polymer therapeutics, Nat Rev Drug Discov 2, 347-360 (2003).Wagner, et al., The emerging nanomedicine landscape, Nat Biotechnol 24,1211-1217 (2006). Scheinberg, et al., Conscripts of the infinite armada:systemic cancer therapy using nanomaterials, Nat Rev Clin Oncol 7,266-276 (2010). Miyata et al., Polymeric micelles for nano-scale drugdelivery, React. Func. Polym. 71, 227-234 (2011). Lee et al.,Multifunctional mesoporous silica nanocomposite nanoparticles fortheranostic applications, Acc Chem Res 44, 893-902 (2011). Schroeder etal., Treating metastatic cancer with nanotechnology, Nat Rev Cancer 12,39-50 (2012). Rosenholm et al., Nanoparticles in targeted cancertherapy: mesoporous silica nanoparticles entering preclinicaldevelopment stage, Nanomedicine (Lond) 7, 111-120 (2012). Ashley et al.The targeted delivery of multicomponent cargos to cancer cells bynanoporous particle-supported lipid bilayers, Nat Mater 10, 389-397(2011). Vivero-Escoto et al., Silica-based nanoprobes for biomedicalimaging and theranostic applications, Chem Soc Rev 41, 2673-2685 (2012).Tada et al., In vivo real-time tracking of single quantum dotsconjugated with monoclonal anti-HER2 antibody in tumors of mice, CancerRes 67, 1138-1144 (2007). Yet, despite extensive particle developmentsto date, no inorganic fluorescent particle imaging probe hassuccessively made the transition to the clinic as a targetedmultifunctional platform technology. Altinoglu et al., Near-infraredemitting fluorophore-doped calcium phosphate nanoparticles for in vivoimaging of human breast cancer, ACS Nano 2, 2075-2084 (2008).Hilderbrand et al., Near-infrared fluorescence: application to in vivomolecular imaging, Curr Opin Chem Biol 14, 71-79 (2010). Choi et al.,Design considerations for tumour-targeted nanoparticles, Nat Nanotechnol5, 42-47 (2010). He et al., Near-infrared fluorescent nanoprobes forcancer molecular imaging: status and challenges, Trends Mol Med 16,574-583 (2010).

Here we describe that a first, ˜7-nm fluorescence nanoparticle, modifiedas a hybrid (optical/PET) targeted platform by the attachment ofradiolabels and cyclic arginine-glycine-aspartic acid-tyrosine (cRGDY(SEQ ID NO: 1)) peptide, is not only well-tolerated in metastaticmelanoma patients, but may preferentially detect and localize presumedintegrin-expressing tumors in a microdosing regime with highsignal-to-noise ratios. No significant serum protein binding isobserved, and the integrity of the particle and its surface componentsare maintained in vivo. Results on safety, pharmacokinetics, dosimetryand targeting suggest general utility of this renally-excreted particlein cancer diagnosis and for potentially guiding treatment planning. Thisdual modality probe constitutes a platform that can be tailored tospecific tumor types which may improve optical and/or PET-based lesiondetection and cancer staging in humans, as well as drug delivery,potentially leading to better and more personalized cancer care.

We used an ultra-small (˜7-nm diameter), dual-modality (optical/PET)inorganic nanoparticle probe for targeted molecular imaging of α_(v)β₃integrin-expressing cancers. This nanoparticle probe is the firstFDA-investigational new drug of its class and properties approved forfirst-in-human studies (FIG. 28A). The fluorescent silica nanoparticle(Cornell, or C dots) covalently sequesters dye molecules in a coreencapsulated by a silica shell to prevent dye leaching and to enhancebrightness and photostability. Ow, et al. Bright and stable core-shellfluorescent silica nanoparticles, Nano Lett 5, 113-117 (2005). Burns, etal. Fluorescent silica nanoparticles with efficient urinary excretionfor nanomedicine, Nano Lett, 9, 442-448 (2009). Absorption-matchedspectra demonstrate this per dye brightness enhancement as differencesin intensity between the emissions of the encapsulated and free dyes(FIG. 28B). A neutral layer of surface poly(ethylene glycol) (PEG)chains enables attachment of a small number of cyclicarginine-glycine-aspartic acid-tyrosine (cRGDY (SEQ ID NO: 1)) peptidesfor potent and selective integrin receptor binding. Benezra, et al.Multimodal silica nanoparticles are effective cancer-targeted probes ina model of human melanoma, J Clin Invest 121, 2768-2780 (2011).Activated integrins induce many structural and signaling changes withinthe cell, in addition to regulating differentiation pathways, mediatingadhesion properties, and promoting cell migration, survival, and cellcycle progression. Hood et al., Role of integrins in cell invasion andmigration, Nat Rev Cancer, 2, 91-100 (2002). The attachment of nuclearimaging labels, such as ¹²⁴I, via tyrosine residues amplifies signalsensitivity for serial PET imaging. The final product, a highlybiocompatible and biostable tumor-selective particle tracer(¹²⁴I-cRGDY-PEG-C-dots), has previously provided a read-out of integrinreceptor status by PET imaging in human melanoma xenografts, thusdefining a distinct class of renally-cleared theranostic platforms fornanomedicine. Jokerst et al., Molecular imaging with theranosticnanoparticles, Acc Chem Res, 44, 1050-1060 (2011).

A first-in-human clinical trial was initiated, employing PET toquantitatively assess the time-dependent tumor uptake andbiodistribution, radiation dosimetry, and safety of this agent in acohort of five patients with metastatic melanoma. Implicit in therationale of using PET imaging is the preclinical evidence that theparticle radiotracer has no pharmacologic, radiogenic, or otherdemonstrable biologic effect. Collins, J. M. Phase 0 clinical studies inoncology, Clin Pharmacol Ther, 85, 204-207 (2009). Kummar et al., Phase0 clinical trials: recommendations from the Task Force on Methodologyfor the Development of Innovative Cancer Therapies, Eur J Cancer, 45,741-746 (2009). Following a single dose intravenous (i.v.) injection ofapproximately 185 megabequerels (MBq) (˜3.4-6.7 nanomoles, nmol) of the¹²⁴I-cRGDY-PEG-particle tracer (specific activity range 27.8-57.4GBq/μmol) into human subjects (FIG. 28A), three whole-body PET-CT scanswere acquired over a 72-hour period to assess pharmacokinetics, inaddition to analyzing metabolites in blood and urine specimens over atwo week interval by gamma counting and radio thin layer chromatography(radioTLC).

Five patients had no adverse events and the agent was well toleratedover the study period. Pharmacokinetic behavior, expressed as thepercentage of the injected dose per gram of tissue (% ID/g), versus timepost-injection and the corresponding mean organ absorbed doses (FIG.28D), were comparable to those found for other commonly used diagnosticradiotracers. Serial PET imaging of this representative patient (FIG.28D) showed progressive loss of presumed blood pool activity from majororgans and tissues, with no appreciable activity seen by 72-hourpost-injection (p.i.). Whole-body clearance half-times in these patientswere estimated to range from 13-21 hours. Interestingly, there was nonotable localization in the liver, spleen, or bone marrow, in contrastto many hydrophobic molecules, proteins, and larger particle platforms(>10 nm). Although patients were pretreated with potassium iodide (KI)to block thyroid tissue uptake, a higher average absorbed thyroid dosewas obtained in this patient relative to other tissues. Particles werealso primarily excreted by the kidneys, with both kidney and bladderwall (after thyroid and tumor, see below), demonstrating one of thehighest % ID/g values by 72 hrs p.i. (FIG. 28D); as is often the casefor renally excreted radiopharmaceuticals, the bladder wall received ahigher average absorbed dose than other major organs and tissues. Thedetailed clinical protocol (FIG. 28C) and the rationale for its design,is further described in Materials and Methods.

These findings highlight the fact that renal, rather than hepatobiliary,excretion is the predominant route of clearance from the body. Efficientrenal clearance will be contingent upon the design of ultra-smallparticle-based platforms or macromolecular systems that are on the orderof the effective renal glomerular filtration size cutoffs of about 10 nmor less. Choi, et al., Targeting kidney mesangium by nanoparticles ofdefined size, Proc Natl Acad Sci USA, 108, 6656-6661 (2011). A smallfraction of the administered activity (less than 5%) was seen as uptakein the stomach and salivary glands of this patient, consistent with freeiodine, which was progressively cleared over the imaging period. Theparticle does not exhibit properties typical of reticuloendothelialagents (i.e., technetium-99m sulfur colloid), whose uptake reflectsmacrophage function, principally in liver. Based on prior data acquiredfor cRGD radiotracers, no unexpected foci of activity were observed.Haubner, R., et al. Synthesis and biological evaluation of a(99m)Tc-labelled cyclic RGD peptide for imaging the alphavbeta3expression, Nuklearmedizin, 43, 26-32 (2004).

Importantly, these properties point to the rather unique pharmacokineticbehavior exhibited by this inorganic dual-modality imaging particle as arenally cleared agent. Metabolic analyses of blood (FIG. 29A) and urine(FIG. 29B) specimens by gamma counting revealed at least an order ofmagnitude drop in tracer activity over a 72-hour period, withessentially no activity remaining at the end of this interval. Particleactivity was largely confined to the blood plasma fraction withoutevidence of significant serum protein binding (data not shown). RadioTLCanalyses of plasma samples revealed a single peak through 24 h p.i.(FIGS. 29C-29E), corresponding to the intact radiolabeled nanoparticle.In urine specimens, two peaks, one corresponding to the intactnanoparticle and the other to a more mobile species (identified as freeiodine), were seen over a 24-hour period (FIGS. 29F-29H). RadioTLCanalyses of the particle tracer (FIG. 29I), radioiodinated peptide(¹³¹I-cRGDY, FIG. 29J), and free radioiodine (¹³¹I, FIG. 29K) confirmedthat the first and second peaks in the radiochromatograms correspondedto the intact nanoparticle and free iodine, respectively. Thus, thesefindings suggested that no measurable loss of particle integrityoccurred over the course of the study, even after excretion through thekidneys.

Although tumor detection was not the goal of this PET microdosing study,surprisingly, despite the low injected dose, there was evidence of tumorlocalization of the particle tracer. In the first case, a whole-bodyPET-CT scan was acquired four hours after i.v. administration of¹²⁴I-cRGDY-PEG-C-dots in a patient with anorectal mucosal melanoma. Oncoronal CT images (FIG. 30A), a large rounded area of decreased density(arrowhead) was seen in the inferior left lobe of the liver, the site ofa known metastatic lesion by prior fluorodeoxyglucose (¹⁸F-FDG) PET/CTimaging (data not shown). On the coronal PET (FIG. 30B) andco-registered PET and CT scans (FIG. 30C), a rim of higher uptakecircumscribed this lesion (FIG. 30B arrowhead; FIG. 30C) andsubsequently cleared by the time of the 24-hour PET scan, suggestingsome preferential localization in this presumed integrin-expressingmetastasis. Significant activity was seen within the bladder,gastrointestinal tract (stomach, intestines), heart, and thegallbladder. In a second subject, a well-defined cystic lesion was seenin the right anterior lobe of the pituitary gland by axial (FIG. 31A)and sagittal (FIG. 31B) magnetic resonance imaging (MRI).

This lesion, a stable finding on prior MM scans, was presumed to be apituitary microadenoma, an intracranial neoplasm known to exhibitmalignant properties, such as neoangiogenesis and progression intoperitumoral tissues. Precise co-registration of this tracer-avid focuswith multiplanar MRI (FIGS. 31C, 31D) and CT (FIG. 31E, 31F) imagesconfirmed its location within the anterior pituitary gland. Initiallyseen as a focus of intense activity, it progressively increased inintensity over a 72-hour interval (arrow, FIGS. 31G-31I), accompanied bya corresponding decrease in surrounding background marrow signal, thusyielding higher tumor-to-background ratios (i.e., tumor-to-brain (TB)˜6) and tumor-to-liver (T/L) ˜2)) (FIG. 31J). PET imaging results may beexplained on the basis of findings in a prior study showing increasedαvβ3 integrin-expression in the parenchyma of a subset of adenomas, aswell as enhanced integrin expression levels in adenomatous stromal cellsin relation to normal connective tissue cells. Farnoud, et al.,Adenomatous transformation of the human anterior pituitary is associatedwith alterations in integrin expression, Int J Cancer, 67, 45-53 (1996).

Our initial data support the notion that human PET studies with thistargeted imaging vehicle are a rationale approach towards detection andlocalization of presumed integrin-expressing tumors. We are planningdose escalation methods to determine an optimal balance among safety,whole-body clearance, and tumor targeting efficiency in more advancedclinical trials. Such PET-driven studies may also permit accuratequantification of integrin receptor expression levels for achievingmaximum targeting efficiency, as well as detection of alterations inthese levels. Further, the use of these quantitative molecular imagingtools can yield information on time-dependent changes in particle uptakeand accumulation within tumors. Kelloff, G. J., et al., The progress andpromise of molecular imaging probes in oncologic drug development, ClinCancer Res, 11, 7967-7985 (2005). For the case of the pituitary adenoma,we were able to compute the cumulative uptake of particles within thislesion. Specifically, we computed the fraction of the total injectedactivity and the number of particles that accumulated at this site overa 72-hour imaging period. Using the measured maximum standardized uptakevalue (SUV_(max), see Methods) of the lesion (i.e., 46.5) at 72 hourspost-injection (nearly a factor of ten higher than that in normalpituitary tissue), corrected for partial volume effects, as well as theapproximate mass of the lesion (i.e., product of the lesion volume andan assumed density of 1 g/cm³), and the patient's body mass, we foundthat, relative to an injected particle load of 2×10¹⁵, roughly 1.78×10¹¹particles (0.01% of the injected dose, % ID) or 1 part per 10,000accumulated at the lesion site. The standard uptake values (SUV) isdefined as the activity per gram of tissue divided by the administeredactivity per gram of body mass. Time-dependent changes in the % ID/g anddosimetry of this lesion, in relation to major organs and tissues, areshown in FIGS. 28C and 28D.

The results suggest that the systemically injected particle tracer waswell-tolerated and safe. Safety measures included monitoring of uptakein normal organs, as well as laboratory toxicity indicators. Secondaryobjectives were to estimate radiation doses and assess plasma and urinemetabolic activity. Safety assessments were based on dosimetry, lack ofclinical symptoms, and the absence of any laboratory indications ofparticle (drug) toxicity.

The results obtained from this first-in-human clinical trial point to asystemically injected particle tracer that exhibited favorable andreproducible PK signatures defined by renal excretion. In contrast toreticuloendothelial agents (i.e., technetium-99m sulfur colloid) andmacromolecules, such as antibodies, there was no appreciable particletracer accumulation within the liver, spleen, or bone marrow. Our dataclearly indicate that a large proportion of the administered activitywas eliminated via the urinary system (FIGS. 28E, 29B); activityconcentrations in the urinary bladder were up to an order of magnitudehigher than those in the liver, for example. Based on the conservativeassumption that all hepatic activity is ultimately excreted via thehepatobiliary route, these data are consistent with ˜90% of theadministered activity being excreted via the urinary system and only˜10% via the hepatobiliary route. In remaining organs/tissues, notablyat early time points (2-4 hrs), residual activity largely reflects thatof the blood pool.

Targeted detection did not serve as a study endpoint, and doseescalation procedures to achieve maximum uptake at sites of disease weretherefore not incorporated into the trial design (i.e., no attempt wasmade to adjust particle doses to optimize tumor targeting). However,despite the low nanomolar amounts used, preferential localization andaccumulation of the particle tracer occurred in several tumors. In oneof the patients with a presumed pituitary adenoma, PET imaging resultsshowed progressive net accumulation of particle tracer activity at thesite of the lesion. The results of this study suggest that oursystemically injected particles are well tolerated and exhibit adistinctly unique ‘macromolecular’ PK signature, where bulk renalclearance predominates without significant RES uptake. This is incontrast to many hydrophobic molecules, proteins, and larger-particleplatforms (>10 nm). This feature is highly atypical for nanoparticles(which generally exhibit diameters greater than estimated renal cut-offvalues, and therefore little renal excretion and slower clearance) andwarrants clinical evaluation of our ultrasmall nanoparticle platform.

These results, along with essential data on safety, pharmacokinetics,and dosimetry of the dual-modality C dot imaging platform, suggest thegeneral utility of this human microdosing technique in terms of yieldingkey tumor-specific read-outs for cancer diagnostics. Such estimatedtumor-accumulated particle tracer loads (or concentrations), along withknowledge of cellular inhibitory response (i.e., IC₅₀ or 50% inhibitoryconcentration), can potentially be used to predict therapeutic dosingrequirements for a given drug.

Methods

Synthesis and Characterization of cRGDY-PEG-C-Dots.

For details regarding the synthesis and characterization of PEGylatedand cRGDY (SEQ ID NO: 1) (Peptides International, Louisville Ky.)surface-functionalized fluorescent core-shell silica nanoparticles(cRGDY-PEG-C-dots) encapsulating the organic dye, Cy5 (emission maxima˜650 nm, 2 dye equivalents within the particle core), see an earlierpublication of this group and references therein. Benezra, M., et al.,Multimodal silica nanoparticles are effective cancer-targeted probes ina model of human melanoma, J Clin Invest, 121, 2768-2780 (2011). Inbrief, particles were prepared by a modified Stober-type silicacondensation. Bogush, et al., Preparation of monodisperse silicaparticles: Control of size and mass fraction, J Non-Cryst Solids, 104,95-106 (1988). Herz, et al., Large stokes-shift fluorescent silicananoparticles with enhanced emission over free dye for single excitationmultiplexing, Macromol Rapid Commun, 30, 1907-1910 (2009). Sadasivan, etal., Alcoholic solvent effect on silica synthesis—NMR and DLSinvestigation, J Sol-Gel Sci Technol, 12, 5-14 (1998). Bifunctional PEGswere derivatized with silanes for attachment to the silica surface andfor peptide coupling. cRGDY peptides (SEQ ID NO: 1) containing thesequence cyclo-(Arg-Gly-Asp-Tyr (SEQ ID NO: 1)) and bearing cysteineresidues (Peptide International) were attached to functionalized PEGchains via a cysteine-maleimide linkage. Hydrodynamic radius,brightness, and concentrations of cRGDY-PEG-Cdots, as against free Cy5dye, were analyzed on a Zeiss LSM 510 Confocor 2 FCS using HeNe 633-nmexcitation.

Radiolabeling of cRGDY-PEG-C-Dots.

Tyrosine residues were conjugated to PEG chains for attachment ofradioiodine moieties (i.e., ¹²⁴I, ¹³¹I). Hermanson, G, BioconjugateTechniques, (Academic Press, London, U K, 2008). Yoon, T. J., et al.Specific targeting, cell sorting, and bioimaging with smart magneticsilica core-shell nanomaterials. Small, 2, 209-215 (2006). Radiolabelingof cRGDY-PEG-C-dots was performed using the IODOGEN method (Pierce).Piatyszek, et al., Iodo-Gen-mediated radioiodination of nucleic acids,Anal Biochem, 172, 356-359 (1988). The radiolabeled product was elutedfrom PD-10 columns and assayed using a dose calibrator (Capintec, RamseyN.J.) and radioTLC; specific activities of the ¹²⁴I-bound particlefractions were on the order of 1450 millicuries (mCi)/μmole andradiochemical purity was greater than 95%.

Patient Selection.

Metastatic melanoma subjects with histological confirmation of diseaseand harboring newly diagnosed or recurrent tumor were eligible for thetrial. Individuals who had medical illness unrelated to melanoma, whichwould preclude administration of the particle tracer, were excluded fromthe study. Potassium iodide solution (SSKI, 130 mg per day) wasadministered 2 days prior to and up to 2 weeks after intravenousinjection of the radio-iodinated particle tracer (¹²⁴I-cRGDY-PEG-C-dots,185 MBq/5 ml) to block thyroid function. This protocol was approved bythe Institutional Review Board of the Memorial Sloan Kettering CancerCenter. Patients were tested for hemotologic, renal, and liver functionbefore and after PET and provided signed informed consent.

Image Acquisition and Processing.

Low-dose spiral CT scans were obtained per standard procedure, followedby the acquisition of three whole-body PET scans on a dedicated GEDiscovery STE PET/CT scanner 4, 24, and 72 hours post-injection of theparticle tracer. Positron emission data was reconstructed using theordered subsets expectation maximization (OSEM) algorithm. Images werecorrected for attenuation using the CT transmission data collected overthe same region as for emission imaging, and registration of the serialdata set was performed using CT data sets.

Imaging and Metabolic Analyses.

For pharmacokinetic and dosimetric analyses, regions-of-interest (ROIs)were drawn on PET imaging data (AW Workstation, GE Healthcare,Ridgewood, N.J.) to extract mean and maximum standard uptake values(SUVs) for all major normal organs and tissues, including brain, lung,left ventricle, liver, spleen, intestine, kidneys, bladder, muscle,breast, and tumor(s). For pharmacokinetic evaluation, SUVs wereconverted to % ID/g values (i.e., SUV=% ID/g tissue×patient bodymass/100). Organ/tissue uptake data was supplemented by time-activitydata from the blood and urine. Venous blood and urine specimens werecollected at approximately 30-min, 3-hr, 24-hr, 72-hr, and up to 2 weeksp.i. of ¹²⁴I-cRGDYPEG-C-dots. Following centrifugation of whole bloodspecimens (4000 rpm, 10 min), plasma supernatant, along with urinespecimens, were assayed in a scintillation well counter (1480 AutomaticGamma Counter, Perkin Elmer, Shelton Conn.) calibrated for ¹²⁴I.RadioTLC analyses were additionally performed on biological specimens.RadioTLC analyses of the particle tracer, native peptide (cRGDY (SEQ IDNO: 1)) labeled with ¹³¹I, and free iodine (¹³¹I) served as controls tofacilitate interpretation. Activities (counts per minute, cpm) wereconverted to microcuries, decay-corrected, and adjusted for volumesaliquoted. Final values were expressed as % ID/g. Retention factor(R_(f)) values for the tracer were obtained and used for identificationof the parent compound and possible metabolites.

Radiation Dosimetry.

The standard radiation dosimetry method is an adaptation of thatpromulgated by the MIRD (Medical Internal Radionuclide Dosimetry)Committee, accounting for the physical properties of the administeredradionuclides (¹²⁴I) as well as the biological properties(pharmacokinetics and biodistribution) of the radiopharmaceutical inindividual patients. The ¹²⁴I emissions and their respective frequenciesand energies are obtained from the MIRD Radionuclide Data and DecayScheme publications. Serial whole-body PET scans enabled derivation ofnormal-organ absorbed dose (rad and rad/mCi) estimates using ROI-derivedtime-activity data. PET scans were acquired with all parametersidentical, including the scan time. Using the patient's total-body mass(in kg) and the 70-kg Standard Man organ masses, the total-body andorgan ROI data (i.e., mean standard uptake values (SUVs) were convertedto activities (i.e., fraction of the injected dose). The foregoingimage-derived time-activity data were fit to exponential functions usinga least-squares fitting algorithm and the resulting time-activityfunctions analytically integrated, incorporating the effect of physicaldecay of ¹²⁴I to yield the cumulated activities (or residence times) inμCi-hr/μCi in the organs and total body. Cumulated activities were usedto calculate ¹²⁴I-labeled particle mean absorbed doses to the organs(rad/mCi) and effective dose (rem/mCi) for the 70-kg Standard Mananatomic model by employing the OLINDA EXM MIRD program. Loevinger etal., MIRD Primer for Absorbed Dose Calculations, Society of NuclearMedicine, New York, N.Y., 1991.

Serum Protein Binding Assays.

Whole-blood specimens were collected in serum separator tubes from ametastatic melanoma miniswine model (Sinclair Research Center, MO),followed by centrifugation (4000 rpm, 10 min) to isolate the plasmafraction. An aliquot of serum was set aside for gamma counting; theremaining fraction was treated with ethanol (200 proof, Decon Labs, Kingof Prussia, Pa.), vortexed until cloudy, and placed on dry ice (5 min)to promote precipitation of serum proteins. Following centrifugation,supernatant was collected for gamma counting and the pellet was washedrepeatedly with phosphate buffered saline and centrifuged (4000 rpm, 10min) to collect 100 μL aliquots of supernatant for gamma-counting (1480Wizard 3″).

EXAMPLE 15 Alpha-MSH-PEG-Cy5-Particles (MSH Peptide—Bound Particles)

The N-Ac-Cys-(Ahx)₂-D-Lys-ReCCMSH (or alpha MSH) peptide used fornanoparticle conjugation has the structure shown in FIG. 32. It isattached to the nanoparticle via the N-terminal cysteine thiol. A spacerof two amino hexanoic acid units separates the nanoparticle attachmentpoint from the D-Lys-ReCCMSH targeting molecule. The original ReCCMSHtargeting molecule is shown in FIG. 33. It was designed to targetradionuclide to melanoma tumors for imaging and therapy. The MSH peptideanalog could be directly radiolabeled with ^(99m)Tc or ¹⁸⁸Re (at thesite of the Re) or via a metal chelator appended to the amino terminus.

The alpha MSH peptide analog shown in FIG. 32 is quite different fromthe original MSH analog shown in FIG. 33. The original MSH moleculecould not be attached to a nanoparticle. The nanoparticle MSH peptidecontains a free amino group containing side chain for radiolabeling.This is currently a D-Lysine but could be an amino group terminated sidechain of 1 to many carbons in length.

The conjugation of the N-Ac-Cys-(Ahx)₂-D-Lys-ReCCMSH (or alpha-MSH) tothe nanoparticle allows the nanoparticle to target and bind melanomacells and tumors. The resulting particles, or alpha-MSH-PEG-Cy5-C dotswere about 6-7 nm i.d. using FCS and contained, on average, about 2.6dyes per particle. The number of alpha-MSH ligands per particle wasestimated at <10.

Competitive binding studies using N-Ac-Cys-(Ahx)₂-D-Lys-ReCCMSH (oralpha-MSH) conjugated particles: In a competitive binding assay with amelanocortin-1 receptor agonist (¹²⁵I-NDP), the alpha MSH conjugatednanoparticles had an IC₅₀ for cultured B16/F1 melanoma cells of6.6×10⁻¹⁰ M (FIG. 34A), while a scrambled sequence version of themolecule had an IC₅₀ of 2.3×10⁻⁷ M (FIG. 34B). In addition, there was a3 order of magnitude difference in binding. For reference in the sametype of competitive binding assay, the original DOTA-ReCCMSH had an IC₅₀of 2.1×10⁻⁹ M.

The N-Ac-Cys-(Ahx)₂-D-Lys-ReCCMSH (or alpha-MSH) peptide-boundnanoparticle conjugate had better affinity for the B16/F1 cells than theoriginal DOTA-ReCCMSH and much larger affinity than the scramble peptidenanoparticle.

Dose-response data was additionally obtained as a function of targetedparticle concentrations (FIG. 35A) and incubation times (FIG. 35B) forboth B16F10 and M21 melanoma cell lines. Saturation concentrations forthese lines, based on flow cytometry studies, were found to be on theorder of ˜400 nM for these two cell types, and at least 2 hr incubationtimes were needed to maximize binding.

Human M21 cell survival studies, performed over a range of particleconcentrations for a fixed incubation time of 48 hr demonstrated nosignificant loss of cell viability (FIG. 36).

¹²⁵I-radiolabeled alpha-MSH conjugated nanoparticles demonstrated bulkrenal excretion over a 24 hr period in both B16F10 and M21 murinexenograft models (FIGS. 37A, 37B); no appreciable particle tracer wasmeasured at later time points (i.e., >24 hrs). In addition, neitherB16F10 nor M21 xenograft models showed significant accumulation of thetargeted particle probe in the reticuloendothelial system (i.e., not anRES agent), nor in the kidney (FIGS. 38A, 38B), the latter organtypically a site that accumulates alpha-MSH non-specifically given itsnet positive charge. Thus, the attachment of alpha-MSH to the particleprobe significantly improved its renal clearance properties andeliminated accumulation within the kidneys.

EXAMPLE 16 Integrin-Mediated Signaling and Modulation of Tumor Biology

Integrin signaling regulates diverse functions in tumour cells,including adhesion/spreading, migration, invasion, proliferation andsurvival. In several tumor types, including melanoma, the expression ofparticular integrins correlates with increased disease progression anddecreased patient survival. Integrin-mediated adhesive interactions haveidentified novel integrin functions in cell survival mechanisms and inthe activation of divergent signaling pathways. It is well known thatbinding of peptides (or clusters of peptides), containing the RGDsequence, to integrin receptors leads to cross-linking or clustering ofintegrins which, in turn, modulates the above processes. It is notclear, however, whether nanoparticles bearing multiple RGD peptideligands can additionally trigger such integrin signaling events, as thiswill reflect a complex dependency on multiple particle-based factors(size, charge, composition, surface chemistry, ligand number/type),tumor (or endothelial) cell type, cell receptor density, and particledosing. Our dose-response studies demonstrate modest activation ofdivergent signaling pathways upon cell exposure to particleconcentrations greater than 100 nM which, in turn, promote M21 and HUVECcell migration, proliferative activity, and alter adhesion properties.

Binding of cRGDY-PEG-C-Dots to M21 and HUVEC Cells

The α_(v)β₃ integrin receptor plays a key role in vascular remodelingand angiogenesis, demonstrating increased expression on endothelial andtumor cell surfaces for a variety of tumor cell lines. This leads to theuse of this receptor as a target for diagnostic and therapeuticpurposes. In our case, the cRGDY-PEG-C dots were tested for theirability to bind to melanoma cells (M21) or to human umbilical vascularendothelial cells (HUVEC) as a function of concentration and time.Initial dose-response studies showed a progressive increase in thebinding of cRGDY-PEG-C dots to M21 and HUVEC cells as a function ofconcentration by flow cytometry (FIGS. 39A-39D, FIG. 47A). Particlebinding demonstrated saturation at about 100 nM for both cell lines,with mean % gated values of about 80% for M21 cells and 96% for HUVECcells. Dose-response behavior was additionally investigated as afunction of particle incubation times for both cell types afterincubating with 100 nM cRGDY-PEG-C dots. Maximum binding was observed at2 hours post-incubation, remaining relatively constant thereafter (FIG.47B).

Endocytosis and Intracellular Trafficking of cRGDY-PEG-C-Dots

To elucidate the nature of the pathway/s utilized by cRGDY-PEG-C-dotsfollowing their incubation in M21 cells—whether this is an α_(v)β₃integrin receptor-mediated and/or non-specific uptake process, weexamined the temperature-dependent uptake of these particles for a 4hour incubation time at three temperatures: 4°, 25° C. and 37° C. Theresults, summarized in FIGS. 39A, 39C indicate an increase incell-associated cRGDY-PEG-C-dots at 37° C. as compared to 25° C. or to4° C. in both cells line tested. Moreover, internalization ofcRGDY-PEG-C-dots was partially blocked in the presence of excess (×250)antibody to α_(v)β₃ receptor in M21 and HUVEC cells (FIGS. 39A, 39C),suggesting a component of receptor-mediated binding. Additionally,uptake in α_(v)β₃-negative M21L cells was roughly a factor of 4- to8-fold lower than that seen with α_(v)β₃-expressing cells at both 37° C.and 25° C. (FIG. 39B), respectively.

To further characterize the cellular compartments involved incRGDY-PEG-C dot internalization, we performed colocalization assays inM21 cells with cRGDY-PEG-C dots and biomarkers of different endocytoticvesicles. Internalization of the targeted particle (˜1 μM, red, 4-hrincubation) was sensitively detected by an inverted confocal microscope(Leica TCS SP2 AOBS) equipped with a HCX PL APO objective (63×1.2 NAWater DIC D) (FIG. 39D). Using endocytotic markers LysoTracker Red (100nM, green) (FIG. 39D) and transferrin-Alexa488, uptake into acidicendocytic structures was confirmed, suggesting clathrin-dependentpathway activity and gradual acidification of vesicles. FIG. 39D showsco-localization data between the particle and acidic endocytic 30vesicles (yellow puncta). Uptake into macropinocytes was also observedwith 70-kDa dextran-FITC which co-localized with cRGDY-PEG-C dots; thisfinding suggested a second pathway of internalization. Nuclearcounterstaining (blue) was done with Hoechst. No particles entered thenucleus. Time lapse imaging confirms particle uptake into M21 cells andco-localization with the lysosomal marker, Lamp1.

Competitive α_(v)β₃ Integrin Receptor Binding and Molecular Specificity

Competitive binding assays showed blocking of receptor-mediated particlebinding in M21 and HUVEC cells (FIG. 48A) by 80%-85% in the former and30-40% in the latter using excess (×50-×100) cRGDY peptide (SEQ IDNO: 1) and gamma counting of the radiolabeled particle tracer(124I-PEG-cRGDY-C dots). By contrast to cRGDY-PEG-C dots, significantreductions were seen in the magnitude of receptor binding in M21(˜30%-43%) and HUVEC (˜13%-27%) cells after incubation with non-targeted(i.e., PEG-C dots) particle probes by flow cytometry (FIG. 48B).

Influence of cRGDY-PEG-C Dots on Cell Viability and Proliferation

To demonstrate that cRGDY-PEG-C dots did not adversely affect cellsurvival and proliferation, G0/G1 phase-synchronized M21 and HUVEC cellswere exposed to a range of particle concentrations (25-200 nMcRGDY-PEG-C dots; 15 min or 24 hr) and incubation times (0-93 hrs) inserum-supplemented media (2%, 10% FBS) at 37° C., and time-dependentchanges in absorbance were measured using an optical plate reader (λ=440nm). Relative to controls (i.e., serum-supplemented media), absorbancemeasurements were seen to be relatively constant over the range ofparticle concentrations tested, suggesting no significant loss of cellsurvival (FIGS. 49A, 49B). Further, no time-dependent decreases inabsorbance were found following multi-dose (n=5) addition of 100 nMcRGDY-PEG-C dots to M21 and HUVEC cells, suggesting no alteration in theproliferative properties of cells (FIGS. 49C, 49D).

Activation of the FAK pathway by cRGDY-PEG-C-dots

The binding of a ligand to the α_(v)β₃ integrin receptor is known toinitiate signal transduction pathways. Upon the binding, integrinclustering occurs which leads to autophosphorylation of the non-receptorkinase FAK at tyrosine 397, a binding site for the Src family kinases.Recruitment of Src kinase results in the phosphorylation of FAK attyrosine 576/577 and FAK at tyrosine 925 in the carboxyl-terminalregion. The phosphorylation of FAK at tyrosine 397 is also known toactivate numerous signaling cascades and downstream pathwayintermediates, such as the Ras-Mitogen Activated Protein (MAPK)signaling, which induces activation of Raf/MEK/Erk andphosphatidylinositol 3-kinase (PI3K)/AKT pathways (AKT, mTOR, S6K) (FIG.40A).

To determine whether α_(v)β₃ integrin-binding cRGDY-PEG-C-dots modulatesthe activity of these pathways, we treated serum-deprived (0.2% FCS) M21cells with 100 nM of cRGDY-PEG-C-dots for 2 hrs at 37° C.;serum-deprived cells treated with 0.2% FCS alone served as controls.Western blot analyses of lysates from particle-exposed cells revealedenhancement of the phosphorylation levels of multiple proteinintermediates: pFAK-397 and pFAK-576/577, Src, pMEK, pErk, and AkT (FIG.40B), which suggested activation of downstream signaling pathways. Thesefindings, depicted graphically as normalized intensity ratios, have beenexpressed relative to their respective total protein levels, andnormalized to corresponding values measured under control conditions(FIG. 40C). Incubation of cells with PEG-C dots did not augmentphosphorylation levels of the proteins tested (data not shown).

We evaluated whether the observed activation of downstream signalingpathways was dependent on the phosphorylation of FAK at tyrosine 397 byblocking this pathway with a small molecule inhibitor, PF-573228. Thisinhibitor interacts with FAK in the ATP-binding pocket and effectivelyblocks both the catalytic activity of FAK protein and subsequent FAKphosphorylation on Tyr³⁹⁷. Following the addition of two concentrationsof PF-573228 to serum-deprived M21 cells previously exposed to 100 nMcRGDY-PEG-C dots, Western blot analyses revealed inhibition of thephosphorylation of FAK on Tyr³⁹⁷ and Src (FIG. 41A), graphicallydisplayed in FIG. 41B. Inhibition of MEK or Erk phosphorylation wasobserved only at the higher inhibitor concentration (FIGS. 41A, 41B).

Effect of cRGDY-PEG-C Dots on Cellular Migration

The α_(v)β₃ integrin receptor, which is highly expressed on many typesof tumor and angiogenic cells, is known to modulate a number ofdownstream biological processes, including cell migration, adhesion,proliferation, invasion, and angiogenesis. In the following experiments,we sought to determine whether cRGDY-PEG-C dots alter the migration ofM21 and HUVEC cells. An initial set of experiments examinedtime-dependent changes in M21 cell migration, as reflected in mean areasof closure, using time-lapse imaging at three successively higherparticle concentrations (0 nM-400 nM; 37° C.) during a 96-hour timeperiod (FIG. 42A, panels i-xx). Statistically significant increases inmean area closure were observed over a 96-hr period, as a percentage ofthe baseline values (t=0), which were relatively constant for theparticle concentration range used (i.e., ˜87%-92%), as compared tocontrol samples (73%; p<0.05) (FIG. 42A, 42B). No significant changeswere seen at earlier time intervals (FIG. 42B).

Incubation of HUVEC cells (37° C., 20 hours) with a range of cRGDY-PEG-Cdot concentrations of (100-400 nM) in the presence of 0.2% FCS showed astatistically significant increase in the mean area closure for aconcentration of 400 nM (i.e., 34%) (FIGS. 43A, 43B), as compared to 19%(p<0.05) for non-particle exposed cells. No appreciable change inmigration was observed for the lower particle concentrations used (FIGS.43A, 43B). Particle-exposed cells were seen to exhibit higher migrationrates as compared to control cells.

We further showed that increases in cell migration rates were attenuatedby the addition of FAK inhibitor, PF-573228. Initial phase contrastimages were acquired after incubating HUVEC cells with 400 nM particlesover a 24 hour time interval (FIG. 44A, panels i-viii), and mean areaclosure was determined and graphically displayed relative to serum alone(FIG. 44B). Percentage change in mean areas of closure relative tocontrols, and before and after addition of an inhibitor, was seen todecrease from +34% to +3%. Statistically significant differences werefound between values for particle-exposed cells without inhibitor(p<0.03) and particles treated with different inhibitor concentrations(250 nM, 500 nM; p<0.001) relative to serum-deprived controls;differences between the first two groups were also statisticallysignificant (FIG. 44C).

Effect of cRGDY-PEG-C Dots on Cellular Adhesion and Spreading

The RGD tripeptide is known to be a component of extracellular matrixconstituents, fibronectin and vitronectin. A competitive binding assaywas performed using M21 cells (10⁴ cells/well) pre-incubated (25° C., 30minutes) with or without 400 nM cRGDY-PEG-C dots and fibronectin, aftertransferring cells to fibronectin-coated wells. About ˜60% of the cellsthat were not pre-incubated with cRGDY-PEG-C-dots attached and spread onfibronectin coated plates in the first 30 minutes. By contrast, a verylow percentage (15%) of M21 cells pre-incubated with 400 nMcRGDY-PEG-C-dots attached and spread on the fibronectin coated well,even 120 minutes after seeding (FIGS. 45A, 45B). Average cell counts of‘elongated’ (spreading cells) versus ‘rounded’ cells over a 2 hourperiod revealed that in the absence of particles (i.e., controlcondition), cell spreading (elongated′ cells) was observed in themajority of cells, while a predominantly ‘rounded’ appearance was seenin the case of particle-exposed cells (FIG. 45A). These results aregraphically depicted in FIGS. 45B and 45C, respectively. Quantificationof the optical densities of cells after addition of methylene blue(absorbance) revealed that cells which attached and spread onfibronectin (i.e., no particle pre-incubation) took up two times moremethylene blue than particle-exposed cells (FIG. 45D). We observed thesame phenomena if vitronectin-coated wells were used (data not shown).Altogether, these data demonstrate that cRGDY-PEG-C-dots enhanced themigration and spreading of cells through the binding to the integrinα_(v)β₃ receptor found on M21 and HUVEC cells.

Influence of cRGDY-PEG-C Dots on Cell Cycle in M21 Cells

Since integrin receptors are involved in survival and proliferation ofthe cells, we analyzed the effect of cRGDY-PEG-C-dots on cell cycle.G₀/G₁-phase-synchronized M21 cells were incubated for 48 hours using twoconcentrations of cRGDY-PEG-C-dots (100 nM, 300 nM) in 0.2% FCSsupplemented media. Over this range, the percentage of cells in the Sphase rose by 11%, with statistical significance achieved at 100 nM(p<0.05) and 300 nM (p<0.005) in relation to controls (FIGS. 46A, 46B).Corresponding declines of 6% and 5% were seen in the G1 and G2 phases ofthe cell cycle, respectively. Taken together, these data indicate anenhancement in S phase in the presence of cRGDY-PEG-C dots.

Materials and Methods

Reagents, Antibodies and Chemicals.

RPMI 1640, fetal bovine serum (FBS), penicillin, streptomycin and HBSS(without calcium and magnesium containing 0.25% trypsin and 0.05% EDTA)were obtained from the Core Media Preparation Facility, Memorial SloanKettering Cancer Center (New York, N.Y.). Anti-polyclonal rabbit:phospho-Erk1/2 (pErk1/2^(Thr 202/Tyr204)), phospho-AKT(p-Akt^(Ser473))phospho-Src (p-Src^(Tyr419)), phospho-p70 S6(p-p70S6^(Thr389)), phospho-MEK1/2 (p-MEK1/2^(Ser217/221)),phosho-FAK-^(Tyr397), phospho-FAK-^(Tyr576/577) phosho-FAK^(Tyr925) Erk,AKT, Src, p70S6, MEK1/2 (47E6) and FAK were obtained from Cell SignalingTechnology (Danvers, Mass.). Goat anti-rabbit IgG, Goat anti-mouse IgGhorseradish peroxidase (HRP) conjugates were acquired from Santa CruzBiotechnology (Santa Cruz, Calif.). Propidium iodide, RNase A,Transferrin-Alexa Fluor 488 conjugate, FITC-Dextran, Fluorescein,LysoTracker Red DND-99, LysoTracker Green DND-26, pHrodo Red Dextran andHoechst were procured from Invitrogen-Life Technologies (Carlsbad,Calif.). PF-573228 was obtained from TOCRIS bioscience (Ellisville,Mo.). Cyclo (Arg-Gly-Asp-D-Tyr-Cys) was from Peptides International(Louisville, Ky.).

Synthesis of cRGDY-PEG-C Dots and PEG-Dots.

Fluorescent particles, containing the organic dye Cy5, were prepared bya modified Stober-type silica condensation, as previously described.Tyrosine residues were conjugated to PEG chains for attachment ofradioiodine. cRGDY peptides (SEQ ID NO: 1) containing the sequencecyclo-(Arg-Gly-Asp-Tyr (SEQ ID NO: 1)), and bearing cysteine residues(Peptide International), were attached to functionalized PEG chains viaa cysteine-maleimide linkage. The number of cRGD ligands pernanoparticle was empirically calculated.

Mechanism of PEG Attachment to the C Dot Surface.

Bifunctional PEGs, MAL-dPEG®12-NHS ester (Quanta Biodesigns, Ltd) werederivatized with silanes, specifically 3-aminopropyl triethoxysilane(Gelest), for attachment to the silica surface and for peptide couplingvia reactions between the sulfhydryl groups and maleimide moieties ofthe derivatized PEGs. In addition, methoxy-capped PEG chains were addedto the particle surface using functional organosilicon compounds (Sicompounds), specifically (MeO)3 Si-PEG (Gelest), according to modifiedprotocols. Briefly, (MeO)3 Si-PEG was added, at approximately threemolar excess, to particles in a water/alcohol basic mixture (˜1:5 v/v),and the mixture stirred overnight at room temperature.

Hydrodynamic Size and Relative Brightness Comparison Measurements byFluorescence Correlation Spectroscopy (FCS).

The hydrodynamic radius, brightness, and concentrations of cRGDY-PEG-and PEG-dots, as against free Cy5 dye, were initially determined bydialyzing these particle samples to water, diluting into physiologicalsaline (0.15 M NaCl in H₂O), and analyzing the resulting specimens on aZeiss LSM 510 Confocor 2 FCS using HeNe 633-nm excitation. Theinstrument was calibrated with respect to particle size prior to allmeasurements. Average hydrodynamic sizes of the dye and particle specieswere estimated based on diffusion time differences, while relativedifferences in brightness were assessed using count rates permolecule/particle.

Cells and Cell Culture:

Human melanoma cell line M21 and M21 variant M21L (a_(v) negative) wereobtained from D. A. Cheresh (University of California, San Diego,Calif.). Cells were maintained in RPMI 1640 supplemented with 10% fetalbovine serum, 2 mM L-glutamine and Penicillin Streptomycin. HumanUmbilical Vein Endothelial Cells (HUVECs) were obtained from LONZA(Walkersville, Md.) cultured in EGM-2 medium containing 2% FBS, andgrowth factors LONZA.

In Vitro Cell Binding Studies Using Optical Detection Methods:

To assay particle binding for M21, M21-L or HUVEC cells, 24-well plateswere coated with 10 μg/ml collagen type I (BD Biosciences) in PBS,incubated at 37° C. for 30 minutes, and washed once with PBS. Cells(3.0×10⁵ cells/well to 4.0×10⁵ cells/well) were grown to confluency.Differential binding of cRGDY-PEG-C-dots to M21 or HUVEC cells wasevaluated over a range of incubation times (up to 4 hours) and particleconcentrations (10-600 nM) using flow cytometry. After incubation, cellswere washed with RPMI 1640 media/0.5% BSA, detached using 0.25%trypsin/EDTA, pelleted in a microcentrifuge tube (5 minutes at 153 g,25° C.), re-suspended in BD FACSFlow solution (BD Biosciences), andanalyzed in the Cy-5 channel to determine the percentage ofparticle-bound probe (FACSCalibur, Becton Dickinson, Mountain View,Calif.).

Internalization Study:

The experiment was done as above but incubated for 4 hours at threedifferent temperatures: 4° C., 25° C. and 37° C. Co-incubation of excess(×250) cRGDY (SEQ ID NO: 1) anti-human integrin α_(v)β₃fluorescein-conjugated antibody (Millipore, Billerica, Mass.) withcRGDY-PEG-C-dots was used for receptor blocking to assess specificity.Assays were performed with fixed and live cells. For fixed M21 cells,inverted confocal microscopy (Leica TCS SP2 AOBS) equipped with a HCX PLAPO objective (63×1.2 NA Water DIC D) was used and time-lapse imagingwas used to track live M21 cells. Imaging was acquired followingco-incubation of targeted (or control) particles at severalconcentrations with the following dye markers) for 4 hours: 70-kDadextran-FITC conjugate (1 mg/mL, 37° C. for 30 min; Invitrogen) to labelmacropinosomes, Lysotracker Red (100 nM; Invitrogen) to label theendocytotic pathway (i.e., acidic organelles), and transferrin Alexa488(2 μg/mL.

Competitive Binding Studies:

To assay specific binding M21 cells were incubated (25° C., 4 hours)with ¹²⁴I-cRGDY-PEG-C-dots (25 nM) and excess cRGDY peptide (SEQ ID NO:1). Cells were then washed with RPMI 1640 media/0.5% BSA, and dissolvedin 0.5 ml of 0.2 M NaOH. Radioactivity was assayed using a 1480Automatic Gamma Counter (Perkin Elmer) calibrated for iodine-124.

Western Blot (WB):

M21 cells (1×10⁶ cells/six wells plate) were grown in six wells coatedwith collagen (10 mg/ml), and made quiescent by growing underserum-deprived conditions. The medium was then changed to 0.2% FCS, anddifferent concentrations (25-400 nM) of cRGDY-PEG-C-dots were added (37°C., 0.5-8 hours). Cells were rinsed twice in ice cold PBS, collected bytrypsinization, and the pellet re-suspended in lysis buffer (10 mM Tris,pH-8.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100; ACROS organics, NJ), 1%Na deoxycholate, 0.1% SDS, Complete™ protease inhibitors (Roche,Indianapolis, Ind.), and phosphatase inhibitor cocktail Tablet—PhosSTOP(Roche, Indianapolis, Ind.). Lysates were centrifuged (10 min, 4° C.).Protein concentrations were determined by the bicinchoninic acid assay(BCA, Thermo Scientific, Rockford, Ill.). A 50-μg protein aliquot ofeach fraction was separated by 4-12% gradient sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred toa PVDF membrane (Invitrogen, Carlsbad, Calif.). Membranes were blockedwith 5% non-fat dry milk (Bio-Rad, Hercules, Calif.) in Tris BufferedSaline (TBS)-Tween 0.1%, and signal visualized by ECL chemiluminescence(Thermo Scientific, Rockford, Ill.) or Immobilon Western, (MilliporeBillerica, Mass.) after applying primary (1:1000) and secondary(1:2000-1:5000) antibodies.

Cell Cycle Analysis:

G₀/G₁-phase-synchronized M21 cells were incubated for 48 hours using twoconcentrations (100 and 300 nM) of cRGDY-PEG-C-dots after changing themedium to 0.2% FCS. Following trypsinization, cells were centrifuged(1200 rpm, 5 min), and the pellet suspended in PBS, followed by fixationwith 70% ethanol (4° C., 0.5-1 hour). Cells were successivelyre-suspended in 1 ml PBS containing 1% FCS and 0.1% Triton X-100, 200 μlPBS containing 25 μg/ml propidium iodide, and 100 mg/ml RNaseA (4° C.,60 minutes). Cell cycle analysis was performed by flow cytometry,FACSCalibur, (Mountain View, Calif.) and Phoenix Flow MultiCyclesoftware (Phoenix Flow Systems, Inc., San Diego, Calif.).

Cell Proliferation:

Cells were split (1×10⁴ cells/well) in a 96-well plate coated withcollagen as described in the in-vitro cell binding studies. Differentconcentrations of cRGDY-PEG-C-dots were added (25-200 nM) for 24-48hours at 37° C. Then, 20 ml of the proliferation reagent WST-1 (Roche,Indianapolis, Ind.) was added to the plate (37° C., 1 hour). Fordetermination of optical densities, we used a SpectraMax5 micro platereader (Molecular Devices, Sunnyvale, Calif.). Absorbance was measuredat 440 nm.

Migration Assay:

M21 cells: M21 cells were seeded (6×10⁴ cells/well) using a migrationkit (Oris™ Collagen I coated plate, PLATYPUS TEC). Twenty-four hoursafter seeding the cells, stoppers in the plate were removed. Freshculture media (100 μl) supplemented with 0.2% FBS was introduced andcRGDY-PEG-C-dots were added at several concentrations: 25, 100 and 400nM. Every 24 hours thereafter, media was replaced, along with newparticles, over a 72 hr time interval. Prior to incubating the plate at37° C. overnight, time zero images were captured by the Axiovert 200Mmicroscope (Carl Zeiss) using a 5× (0.15 NA) objective and using a scanslide module in the Metamorph software (molecular devices, PA). Serialmicroscopy was then performed and images captured every 24 hrs for atotal of 96 hours post-incubation. The data were analyzed by usingImageJ software. HUVEC cells: HUVEC cells were additionally seeded(5×10⁴ cells/well) and, 24 hours later, incubated with several particleconcentrations (100, 200, and 400 nM) after replacement of the media. Asimilar microscopy procedure was performed as that for M21 cells, withserial imaging acquired 20 hours later.

Adhesion and Spreading Assay:

The effect of cRGDY-PEG-C-dots on the binding of M21 cells tofibronectin coated plates was evaluated by initially coating 96-wellmicro titer plates with fibronectin in PBS (5 μg/ml), followed by 200 μlRPMI/0.5% BSA (37° C., 1 hour,). Cells (1-3×10⁴ cells/100 μl/well) wereincubated (25° C., 30 minutes), with or without 400 nM ofcRGDY-PEG-C-dots in RPMI/0.1% BSA, and added to fibronectin-coated wells(37° C., 30-120 minutes). For quantification of the number of attachedcells, wells were rinsed with RPMI/0.1% BSA to remove non-adherentcells. Adherent cells were fixed with 4% PFA (25° C., 20 minutes) andstained with methylene blue (37° C., 1 hour). The methylene blue wasextracted from cells by the addition of 200 ml of 0.1 M HCl (37° C., 1hour). For determination of optical densities we used a SpectraMax5micro plate reader and absorbance was measured at 650 nm. For spreadingassay: Time lapse was performed (37° C., 2 hours) and images werecaptured by Axiovert 200M microscope (Carl Zeiss) using a 20× (0.15 NA)objective and using a scan slide module in the Metamorph Software(Molecular Devices).

Quantitative Analyses:

In order to quantify the differences in the size and intensity betweenWestern blot bands, we performed densitometry of phosphorylated andtotal protein intermediates using Photoshop CS2 (Adobe, San Jose,Calif.). Bands were scanned at 300 dpi (Scanjet 7650, Hewlett Packard,Palo Alto, Calif.), and converted to grayscale. The lasso tool was thenused to draw a region of interest (ROI) within the boundaries of eachband in order to derive the following: area (number of pixels), meangrayscale value within the selected area (0-255) and the associatedstandard deviation. The product of the first two values for each bandwas computed, and divided by the product for the initial band in eachset (control band), yielding an intensity value for each sample relativeto the control. Finally the ratio of phosphorylated protein to totalprotein and the corresponding propagated error (SD) were computed foreach sample using the relative intensities.

Phase contrast images captured for migration studies were analyzed usingImageJ 1.45 s (National Institutes of Health, rsbweb.nih.gov/ij/) inorder to quantify the extent of cell migration (i.e., area closure) forM21 cells and HUVECs. At high power views, an enclosed area was drawnadjacent to the rim of attached cells seen in each image after stopperremoval. The enclosed area for each image was measured (pixels) and usedto calculate percent closure relative to time zero (following particleaddition and media replacement) as follows: difference in area at agiven time point (24, 48, 72 or 96 hr) and at time zero divided by thesame area at time zero multiplied by 100. The resulting values wereaveraged and a standard error computed for each group.

Statistics:

All graphical values are plotted as mean±SE, except where noted.One-tailed Student's t-test was used to test the statisticalsignificance of differences in cellular migration between HUVECs or M21cells incubated with serum alone or cRGDY-PEG-C dots. One-way analysisof variance (ANOVA) was used to perform statistical pair-wisecomparisons between the percentage of M21 cells in S phase that wereincubated with serum alone, 100 nM or 300 nM cRGDY-PEG-C dots. Weassigned statistical significance for all tests at P<0.05.

EXAMPLE 17 Sphingomyelin Liposomes for Enhanced Tumor Delivery of SilicaDiagnostic/Therapeutic Particles

The therapeutic potential of sub-10 nm particles (e.g., C dots) as drugdelivery vehicles is under investigation. Drug-bound particles have beendeveloped by attaching drugs to particle-bound bifunctionalized PEGchains. Model drugs utilized for this proof-of-concept work are receptortyrosine kinase (RTK) inhibitors. Delivery and accumulation of particlestherapeutic payloads at the target site can be maximized by employingdose escalation strategies to assess for enhanced particle uptake anddistribution within tumors by optical and/or PET imaging. An alternativeapproach to enhance particle load delivery utilizes liposomalformulations to encapsulate therapeutic particle probes. Liposomespassively target tumors via the enhanced permeability and retention(EPR) effect while protecting payloads from degradation in thecirculation. Prior to particle encapsulation, either cRGDY (SEQ IDNO: 1) or linker-drug conjugates will be attached to the particlesurface as an active targeting mechanism to maximize delivery to thetarget site.

Once at the target site, release of particles from liposomalformulations into the tumor interstitium can occur as a result ofupregulated enzymatic systems present in tumors. Extracellular releaseof acid sphingomyelinase (ASMase, acid SMase or SMase) from tumor cellsin response to cellular stress, such as X-ray irradiation and toxins,leads to rapid ceramide-mediated cell injury and, subsequently,apoptosis. Acid SMase hydrolyzes sphingomyelin, present within theliposomal formulations, to ceramide. Ceramide has a role in biologicalsystems as a secondary messenger in the apoptotic process. Ceramide hassignificantly different membrane properties than the parent moleculesphingomyelin. When sphingomyelin-containing liposomes are cleaved withSMase, there is a dramatic change in the membrane rigidity.Sphingomyelin is a suitable lipid for liposome formulation; this is nottrue for ceramide, as its incorporation as a liposome building blockleads to liposome leakage. This leakage will lead to release ofliposomal contents. This opens the possibility for the intracellulartargeting of a large payload of silica nanoparticles, functionalizedwith drugs and/or cell-internalizing markers (i.e., peptide KLAKLAC (SEQID NO: 6) and small molecule inhibitors) to monitor and/or treatdisease.

To determine whether liposome encapsulation significantly increasesparticles delivery to the tumor site relative to non-encapsulatedparticle probes, we will utilize optical and PET imaging approaches tomonitor uptake of both sphingomyelinase liposome encapsulated andnon-encapsulated particle batches in EGFRmt+ expressing brain tumorxenograft models (i.e., H1650, A431) both liposomes and particle probeswill contain optical markers for visualization.

Ref: Sochanik Al, Mitrus I, Smolarczyk R, Cichoń T, Snietura M, Czaja M,Szala S. Experimental anticancer therapy with vascular-disruptivepeptide and liposome-entrapped chemotherapeutic agent. Arch Immunol TherExp (Warsz). 2010 June; 58(3):235-45.

The foregoing methods for detecting and targeting stressed cells involvea formulation comprised of bilayer liposomes each having an initialouter layer of liposome-forming lipids, sphingomyelin, or a mixturethereof; a second inner layer of liposome-forming lipids, sphingomyelinor a mixture thereof; the first and second layers forming a bilayerliposome and defining an interior space therein; wherein the interiorspace contains a silica nanoparticle. The silica nanoparticle and/orliposome will be compromised of optical markers and/or PET labels (dye,fluorophore, radiolabel, contrast agent), an enzyme substrate, and/ortherapeutic agents, including cytotoxic drugs, DNA segments, or aradiotracer indicator label. The marker- and/or drug-labeled silicaparticles, contained within sphingomyelin liposomes, contact a cellsample under conditions wherein sphingomyelinase is present in orreleased by the cell. This enzymatic contact will, in turn, hydrolyzethe sphingomyelin in the liposome to release the silica nanoparticleand/or marker label and/or drug from the hydrophilic (interior) orhydrophobic (bilayer) compartments of the liposome.

The scope of the present invention is not limited by what has beenspecifically shown and described hereinabove. Those skilled in the artwill recognize that there are suitable alternatives to the depictedexamples of materials, configurations, constructions and dimensions.Numerous references, including patents and various publications, arecited and discussed in the description of this invention. The citationand discussion of such references is provided merely to clarify thedescription of the present invention and is not an admission that anyreference is prior art to the invention described herein. All referencescited and discussed in this specification are incorporated herein byreference in their entirety. Variations, modifications and otherimplementations of what is described herein will occur to those ofordinary skill in the art without departing from the spirit and scope ofthe invention. While certain embodiments of the present invention havebeen shown and described, it will be obvious to those skilled in the artthat changes and modifications may be made without departing from thespirit and scope of the invention. The matter set forth in the foregoingdescription and accompanying drawings is offered by way of illustrationonly and not as a limitation.

What is claimed is:
 1. A fluorescent silica-based nanoparticlecomprising: a silica-based core; a fluorescent compound within the core;a silica shell surrounding at least a portion of the core; an organicpolymer attached to the nanoparticle, thereby coating the nanoparticle;and a plurality of D-Lys-ReCCMSH-containing peptide ligands no greaterthan twenty in number attached to the polymer-coated nanoparticle,wherein the polymer-coated nanoparticles with the ligands attachedthereto has a hydrodynamic diameter within a range from 1 nm to 15 nm asmeasured by dynamic light scattering.
 2. The nanoparticle of claim 1,wherein the organic polymer comprises polyethylene glycol.
 3. Thenanoparticle of claim 2, wherein the polyethylene glycol is attached toa silica surface of the nanoparticle via an amino-silane coupled to anactivated ester group on the organic polymer leading to an amide bond.4. The nanoparticle of claim 3, wherein the nanoparticle is coated withmaleimido-terminated polyethylene glycol chains for attachment of theplurality of D-Lys-ReCCMSH peptide ligands.
 5. The nanoparticle of claim1, wherein each of the plurality of D-Lys-ReCCMSH-containing peptideligands is attached to the polymer-coated nanoparticle via a N-terminalcysteine thiol.
 6. The nanoparticle of claim 5, wherein a spacercomprising two amino hexanoic acid units separates the polymer-coatednanoparticle and each of the plurality of D-Lys-ReCCMSH peptide ligand.7. The nanoparticle of claim 1, wherein a plurality ofLys-ReCCMSH-containing peptide ligands no greater than ten in number areattached to the polymer-coated nanoparticle.
 8. The nanoparticle ofclaim 1, comprising a radionuclide, wherein each of the plurality ofD-Lys-ReCCMSH-containing peptide ligands are labeled with theradionuclide.
 9. The nanoparticle of claim 8, wherein the radionuclidecomprises a member selected from the group consisting of ^(99m)Tc, ¹²⁵I,⁹⁰Y, ¹³¹I, ¹⁷⁷Lu, and ¹⁸⁸Re.
 10. The nanoparticle of claim 8, whereineach of the plurality of D-Lys-ReCCMSH-containing peptide ligandscomprise a free amino group containing side chain for radiolabeling. 11.The nanoparticle of claim 10, wherein the free amino group containingside chain comprises D-Lysine or an amino group terminated side chain ofone or more carbons in length.
 12. The nanoparticle of claim 10, whereineach of the plurality of D-Lys-ReCCMSH-containing peptide ligands arelabeled with the radionuclide via a metal chelator appended to the aminoterminus of the plurality of D-Lys-ReCCMSH-containing peptide ligands.13. The nanoparticle of claim 1, further comprising a therapeutic agent.14. The nanoparticle of claim 1, wherein the fluorescent compound isCy5.
 15. The nanoparticle of claim 1, wherein the fluorescent compoundis Cy5.5.
 16. The nanoparticle of claim 1, wherein the plurality ofD-Lys-ReCCMSH-containing peptide ligands is a plurality ofN-Ac-Cys-(Ahx)₂-D-Lys-ReCCMSH-containing peptide ligands.